PT2344539E - Treatment of pediatric acute lymphoblastic leukemia - Google Patents

Description

DESCRIPTION

" TREATMENT OF PEDIATRIC ACUTE LYMPHOBLASTIC LEUKEMIA " The present invention relates to a CD19xCD3 single stranded bispecific antibody construct for use in a method for the treatment, amelioration or elimination of pediatric acute lymphoblastic leukemia (ALL) in a pediatric patient with ALL in need thereof.

With current treatment of ALL in children, event-free survival rates are about 75%. Therefore, relapse is still frequent. The problems in managing ALL relapses are the resistance of leukemic cells and the patients' decreased tolerance to a second series of treatment after they have already received intensive frontline therapy, resulting in a lower remission rate as well as a higher incidence of relapse a lower overall result. Intensified polychemotherapy is therefore currently essential for the induction of a second complete remission. Depending on a number of prognostic factors, remission can be maintained with chemotherapy and cranial irradiation alone or with intensification of stem cell transplantation (Henze G, von Stackelberg A, Relapsed acute lymphoblastic leukemia.

Childhood Leukemias, C-H Pui, editor, Cambridge: Cambridge University Press; 2006, pp. 473-486).

Although a great deal of progress has been made in the treatment of children with acute lymphoblastic leukemia (ALL) (see for example Möricke et al., Blood 111 (2008), 4477; Moghrabi et al., Blood 109 (2007) , 896), ALL relapses are still the fourth most common pediatric malignancy (Gaynon, Cancer 82 (1998), 1387). In particular for patients with a relapse that appears in the bone marrow within the first three years after diagnosis, therapy for these patients is unsatisfactory and allogeneic stem cell transplantation is the only curative pathway to date. A chemorrefractory relapse after transplantation of hematopoietic stem cells (HSCT) is associated with an unfavorable prognosis, although infusions of donor lymphocytes after transplantation (DLI) may induce remissions in a few patients through the induction of a Leukemia Graft effect (GvL) (Loren et al., BMT 41 (2008), page 483). A second HSCT may be an option and some long-term survivors have been described. However, the disease state prior to HSCT has a predictive value for the post-transplant outcome and it is known that patients with morphological disease or persistent MRD levels> 10-4 of leukemic blasts are at a very high risk of relapse with a very unfavorable result (Bader et al., J. Clin. Oncol., 27 (2009), pp. 377-84). Under these conditions, the disease state prior to the second HSCT is of the utmost importance and every effort should be made to obtain another remission. Despite the use of recently introduced chemotherapeutic agents against refractory leukemia (Jeha, Semin. Hematol. 46 (2009), 76-88), patients with relapse after HSCT often have refractory disease and these patients are very susceptible to toxicity related to chemotherapy. For these patients, non-chemotherapeutic and less toxic strategies are needed to induce remission in these patients.

Aware of the drawbacks mentioned above of conventional therapies for pediatric ALL, there is still a need for an improved treatment regimen. The present invention is directed to a construction of a CD19xCD3 bispecific single chain antibody for use in a method of treating, ameliorating or eliminating pediatric acute lymphoblastic leukemia (ALL) in a pediatric patient with ALL in need thereof. This immunological pathway using antibodies that perform involving T cells provides for the first time a non-chemotherapeutic and less toxic treatment for pediatric patients with ALL.

Recently, a significant clinical activity of CD19xCD3 single chain bispecific antibody (blinatumomab) in non-Hodgkin lymphoma and CD19 (NHL) positive Hodgkin's lymphoma was demonstrated in a phase I study, with impressive partial regressions and complete tumors (Bargou et al., Science 321 (2008): 974-7). Anderson et al. (Blood J. 80 (1992): 2826-34) disclose a bispecific heteroconjugate between an anti-CD19 antibody and an anti-CD3 antibody, which is claimed to trigger lysis of acute lymphoblastic leukemia cells.

In a further ongoing clinical trial, treatment with the above mentioned CD19xCD3 single chain bispecific antibody resulted in the elimination of refractory leukemia cells in non-transplanted adult patients positive for MRD suffering from CD19 + ALL.

It has now surprisingly been found in two compassionate uses that said single chain bispecific CD19xCD3 antibody is not only suitable for the treatment of ALL in non-transplanted adult patients but also in the relapsed ALL pediatric (or in children ) refractory to conventional ALL therapy, including chemotherapy and hematopoietic allogeneic gemstal cell (HSCT) transplantation.

In this case, the inventors report on the potent anti-leukemic effect of CD19xCD3 bispecific antibody in two children (hereinafter referred to as patient 1 and 2) with acute precursor B lymphoblastic leukemia (ALL) who had a chemo-refractory relapse after a transplantation of hematopoietic allogeneic stem cells (HSCT) from matched, unrelated and haploidene donors.

Prior to treatment with the single chain bispecific antibody CD19xD3, patient 1 was pretreated with allogeneic HSCT and multiple chemotherapies. However, these conventional pediatric ALL therapies have failed in such a way that the patient relapses repeatedly, resulting in an extremely unfavorable prognosis. As follows, the pediatric ALL patient received 15 micrograms / m2 / 24 h of the single chain bispecific antibody CD19xCD3 as a continuous infusion over five weeks. During antibody treatment, the patient exhibited an expansion of donor-derived CD8 + T lymphocytes without any signs of graft-versus-host disease (GvHD). This T cell expansion was associated with a rapid deletion of the patient's leukemic blasts and 10 days after initiation of the antibody treatment, no blasts could be detected in the patient's bone marrow in addition to the level of detection of minimal residual disease (MRD) of 1 cell in 10,000. The patient remained negative for MRD throughout the treatment with the antibody. The patient underwent a second transplantation of stem cells from their haploident mother 2 weeks after the end of the treatment with the single chain bispecific antibody CD19xCD3 and remained negative for the MRD from that time (state November 2009). Patient 2 was fifteen years of age diagnosed as a Philadelphia chromosome carrier with a B-positive precursor ALL for CD19 in April 2001. After chemotherapy, a HSCT was administered from an identical HLA sibling in October In 2002, a relapse in bone marrow was diagnosed and further remission was achieved with matinib and chemotherapy. He then received a second HSCT from an unrelated HLA donor in October 2004. In March 2008, he was diagnosed with a second relapse and he was treated with small-dose chemotherapy and Dasatinib due to resistance to Imatinib. After further chemotherapy with Clofarabine and Cytosine / Arabinoside, he achieved molecular remission and received a third allogeneic HSCT from his haploident father with 3 of 6 different HLA alleles with a treatment with Dasatinib after transplantation. Due to gastrointestinal bleeding and dilatative cardiomyopathy, Dasatinib was stopped 5 months after transplantation. In April 2009, he was diagnosed with a combined central nervous system (CNS) relapse with 7x109 blasts / L in the CNS and 3% of blasts in the bone marrow. The patient was then treated with Nilotinib, intrathecal chemotherapy and fractional CNS irradiation with 18 Gy. Three months after this treatment, the patient's bone marrow remained positive for MRD at a level of 1.1 x 1CT3 while the CNS appeared to be blastocyst free. An analysis of the chimerism in the peripheral blood revealed complete hematopoiesis derived from the donor, from his haploidentical father.

The patient was then treated with the single agent blinatumomab at 15æg / m2 / day for 4 weeks by continuous infusion without any side effects. A bone marrow aspiration at the end of the treatment revealed complete remission with an undetectable bone marrow MRD of <lxlCT4. Like patient 1, patient 2 showed no signs of GvHD during or after treatment with blinatumomab. He is well today and goes to school.

These data convincingly demonstrate that bispecific antibodies engaging T cells can induce a strong Leukemia Graft (GvL) effect in the absence of GvHD in pediatric patients with relapse and precursor B ALL refractory to therapy following an allogeneic HSCT. Therefore, it is preferred in the treatment of pediatric patients described herein that a construct of a single chain bispecific antibody CD19xCD3 induces a grafting effect against leukemia (GvL). In addition, treatment is well tolerated by pediatric patients. Under these conditions, the methods of this invention provide a surprisingly better treatment option for pediatric acute lymphoblastic leukemia (ALL), especially in those cases in which pediatric ALL is refractory to conventional therapy for ALL, including chemotherapy and / or HSCT allogeneic. Therefore, and in yet another embodiment of the present invention, constructs that will involve T-cells of single stranded bispecific antibodies provided herein (anti-CD19xCD3) may also be employed in pediatric patients who have undergone haematopoietic stem cell transplantation ( allogeneic) (HSCT). As illustrated in this document, even pediatric patients presenting a relapse of their ALL after an HSCT from an unrelated or haploidentified paired donor and / or pediatric patients who were refractory to chemotherapy can be successfully treated by the methods described herein . Exemplified patients such as those documented herein denoted an impressive anti-leukemic response to pharmaceutical intervention as described herein and became MRD negative in the absence of any GvHD (graft versus host disease) signs. Although the survival of children with ALL has improved markedly in recent decades, relapsing ALLs are still the main cause of treatment failure. For patients in the second relapse, allogeneic HSCT is the only curative pathway to date. One of the main anti-leukemic actions of HSCT is the induction of a GvL effect. Unfortunately, the occurrence of GvL is often associated with GvHD, which is still one of the main causes of morbidity and mortality after HSCT. Therefore, the induction of GvL in the absence of a GvHD is a topic of intensive investigation. One route for the induction of a GvL effect is infusion of donor lymphocytes after transplantation (DLI), which is preferably used for the treatment of ALL relapses following an HSCT. Although DLI is very effective in treating CML (Kolb, Blood 76 (1990), 2462), it is less effective in treatment after ALL relapse transplantation (Loren, BMT 41 (2008), 483) and often leads to occurrence of GvHD.

The pharmaceutical and medicinal methods of the present invention provide a novel pathway for the induction of GvL without GvHD. This pathway is the in vivo activation of lymphocytes derived from the donor after HSCT, using small doses of the single chain bispecific antibody CD19xCD3 that binds T cells, directing the T lymphocytes against the CD19 + blasts of the patient's ALL. This antibody denoted impressive anti-lymphoblastic and antileukemic activities in the autologous situation but was never tested in pediatric ALL relapses after HSCT. The two pediatric patients described in the examples denoted relapse of their ALL following a HSCT from matched, unrelated or haploidene donors, and were refractory to chemotherapy. Patients showed an impressive anti-leukemic response after initiation of treatment and became negative for MRD in the absence of any signs of GvHD. The immunological action of the single chain bispecific antibody CD19xCD3 is independent of the presentation of peptide antigen, and this is most likely the reason why despite the extensive in vivo expansion of donor-derived T cells, GvHD was not induced. Small doses of the single chain bispecific antibody CD19xCD3 were sufficient to eliminate the blasts of ALL in the patients, to levels lower than the detection by MRD. Thus, the mode of impairment of the T cells by which this antibody acts is very different from that of the conventional antibodies, which require much larger doses and which fail to compromise the T cells by the Fc part of the antibody molecule due to the absence of Fc in T cells. Through the single chain bispecific CD19xCD3 antibody, highly effective effector T cells can become cytotoxic to ALL blasts without the induction of an alloreactive immune response resulting in GvHD. The inventors decided to carry out a second allogeneic HSCT from a haploident donor in patient 1 after it became negative for MRD. To date (November 2009), this patient continues to be negative for MRD. From initial clinical experience in these two patients with refractory ALL relapses after transplantation, it was concluded that the single chain bispecific CD19xCD3 antibody can induce impressive complete remissions without GvHD. Therefore, treatment with the CD19xCD3 single stranded bispecific antibody in the pre-transplant circumstances for the immunological reduction of leukemia burden and for the treatment of relapses after transplantation are novel therapeutic possibilities for pediatric patients with advanced ALL. The method of the present invention provides the following main advantages: 1. As shown in the following examples, administration of the CD19xCD3 single chain bispecific antibody can be used for the therapy of relapsed pediatric acute lymphoblastic leukemia (ALL) and / or refractory. CD19xCD3 single chain bispecific antibody can replace conventional therapies for pediatric acute lymphoblastic leukemia (ALL), (such as chemotherapy) in pediatric patients not eligible for allogeneic stem cell transplantation, but not only. It can also be used to transform pediatric patients with ALL eligible for such transplantation to a negative condition for MRD. This aspect of the invention is important because MRD negative patients have a lower risk of post-transplant relapse than MRD positive patients. In the best case scenario, pediatric ALL therapy with CD19xCD3 single stranded bispecific antibody renders chemo and / or HSCT superfluous. 2. Pediatric patients with morphological disease or persistent MRD levels of> 1CT4 leukemic blasts are known to be at very high risk of relapse and have prospects for results that are very unfavorable (Bader et al., J. Clin. Oncol. 27 (2009), pp. 377-84). Children treated with the CD19xCD3 single chain bispecific antibody showed an impressive anti-leukemic response following initiation of treatment and became negative for MRD during treatment in the absence of any GvHD signals. The pharmaceutical methods of the invention therefore provide a therapeutic route for the treatment, amelioration or elimination of MRD in pediatric ALL, thereby decreasing or even abolishing the risk of relapse to the patient. The curative potential of allogeneic HSCT depends on the content of MRD before transplantation. Treatment with the single chain bispecific antibody CD19xCD3 can be used to transform the aforementioned high risk pediatric patients to a condition negative for MRD. Thus, the MRD in the refractory ALL of children can be treated by the single chain bispecific antibody CD19xCD3 at the first time. 3. The pharmaceutical methods of the invention show not only a powerful anti-leukemic effect but are also less toxic and cause fewer adverse effects than conventional pediatric therapy for ALL, including allogeneic chemotherapy and / or HSCT. To date, no long-term side effects have been observed following treatment with the CD19xCD3 single chain bispecific antibody. In contrast, conventional pediatric therapies for ALL are very aggressive and are therefore associated with considerable health risks for pediatric patients. In addition, further effects have been reported following conventional therapies for ALL in children (see, for example, Hudson MM, Late complications after leukemia therapy, Childhood Leukemias, CH Pui editor, Cambridge: Cambridge University Press, 2006, pp. , Pp. 750-773; Schmoll, Hoffken, Possinger: Kompendium Internistische Onkologie, S. 2660 ff., 4. Auflage, Springer Medizin Verlag Heidelberg, http://www.cancer.gov/cancertopics/pdg/ treatment / lateeffects / HealthProfessional). In fact, conventional pediatric treatments for ALL use even more aggressive treatment regimens than those used in the therapeutic pathways of ALL treatment regimens in adults. However, as is evident from the relapse rate of about 25% in pediatric ALL, not even these aggressive therapies are sufficient for a final cure of ALL patients. Under these conditions, it is all the more surprising that treatment of refractory ALL and / or relapse of ALL in pediatric patients with the single chain bispecific antibody CD19xCD3 is capable of rendering said patients negative for MRD, providing new therapeutic hypotheses for these patients of high risk. This is a result that could not have been achieved with conventional therapy for pediatric ALL, such as with chemotherapy and / or HSCT. 4. For example, pediatric patients with ALL Ph + (bcr / abl) have a very high risk of relapse, among all ALL subtypes (see below). Although allogeneic HSCT is now considered the treatment of choice in pediatric ALL Ph +, about one third of transplanted patients fall. In addition, as described in more detail below, ALL (&lt; 12 months) newborns present a higher risk of failure of standard or conventional treatment for ALL, the prognosis being more unfavorable than those presenting genetic transpositions MLL (t (4: 1)). The results shown for patient 2 in the following examples and data obtained in the above-mentioned ongoing clinical trial of treatment of adult (non-transplanted) patients with ALL suggest that even ALL Ph + ALL ( bcr / abl) and ALL with MLL genetic transpositions can be successfully treated by administration of the single chain bispecific antibody CD19xCD3. Administration of the CD19xCD3 single chain bispecific antibody therefore provides a novel therapeutic route for pediatric patients with ALL Ph + or ALL and genetic transpositions t (4; 11), in particular for pediatric patients with ALL with minimal residual disease (MRD). This means minimal residual disease (MRD) defined for example by PCR or FACS analysis. Because of their very high cytotoxic activity, only very small doses of the CD19xCD3 single chain bispecific antibody are sufficient for the successful treatment of pediatric ALL, which allows the elimination of leukemia cells even in the bone marrow. 6. Treatment with the single chain bispecific antibody CD19xCD3 has a shorter duration when compared to conventional ALL therapy. Conventional pediatric ALL therapy by chemotherapy generally takes 2 to 3 years, while a very rapid response of pediatric patients to the single chain bispecific antibody CD19xCD3 is observed, as shown in the examples which follow. In addition, the response is long-lasting: patient 1 remained negative for MRD more than 12 months after transplantation; see the following examples. 7. Pediatric patients with relapsed ALL often have chemo-refractory disease and these patients are very susceptible to chemotherapy-related toxicity, a treatment with the single chain bispecific antibody CD19xCD3 provides for the first time a non-chemotherapeutic and less toxic strategy for induce remissions in these patients.

8. As demonstrated in the current clinical trial mentioned above for ALL in adult patients, treatment with the CD19xCD3 single chain bispecific antibody resulted in the elimination of chemo-refractory leukemic cells in non-transplanted adult patients positive for MRD, with ALL CD19 +. While in these patients the cytotoxic T cells in action were derived from the patient, there were no previous data on the use of the single chain bispecific antibody CD19xCD3 after an allogeneic HSCT in a framework involving the mobilization of donor-derived T cells. In the present work, the inventors report for the first time the powerful induction of a GvL effect in the absence of GvHD induced by the single chain bispecific CD19xCD3 antibody mobilizing donor-derived T cells in two pediatric patients with chemo-refractory relapses of ALL CD19 + after a HSCT allogeneic.

In short, treatment with the single chain bispecific CD19xCD3 antibody provides new and improved pediatric ALL therapy, especially for pediatric refractory and / or relapsed ALL.

In a preferred embodiment of the pharmaceutical methods of the invention, acute pediatric or childhood (ALL) lymphoblastic leukemia is pediatric ALL of line B, preferably pediatric acute ALL lymphoblastic leukemia ALL, more preferably pediatric ALL, pre-ALL B ALL, or common ALL (cALL). Even more preferable to pediatric ALL precursor B is ALL common (CALL). The vast majority of cases of pediatric or childhood ALL (> 85%) are of the precursor B cell phenotype (Schultz et al., Blood 109 (2007): 926-935). Since the single chain CD19xCD3 bispecific antibody described herein is directed against the B cell associated marker CD19, said antibody is especially suitable as a therapeutic agent for pediatric acute lymphoblastic leukemia B lineage, more preferably for pediatric ALL of precursor B. The pediatric ALL of precursor B in pediatric ALL, pre-B ALL and pediatric ALL (cALL) (see for example Behm FG, Immunophenotyping, Childhood Leukemias, CH Pui editor, Cambridge:

Cambridge University Press; 2006, pp. 150-209). Acute pediatric lymphoblastic leukemia (ALL), including pediatric acute lymphoblastic leukemia of precursor B and other types of pediatric ALL with B line (cell), and treatments for it are reviewed in Pui CH, Clin. Adv. Hematol. Oncol. 4 (2006): 884-6; Pui CH, Evans WE, N.

Engl. J. Med. 354 (2006): 166-178; Pui CH et al., Lancet 371 (2008): 1030-1043; Pui CH, Jeha S, Nat. Ver. Drug

Discov. 6 (2007): 149-165). More information about pediatric ALL can also be found, for example at http://www.cancer.gov or at http: // www. leukemia lymphoma. org.

From a historical point of view, the Pediatric Oncology Group (POG) and the Children's Cancer Group (CCG) adopted a common set of risk criteria at an international conference supported by the National Cancer Institute (Smith M, et al., J. NCI criteria were based on factors that had international acceptance and reproducibility: age, initial white blood cell (WBC) counts, and presence of disease extramedullary at the time of diagnosis. To further refine therapy, both POG and GCC also used additional risk factors that had been shown to have an impact on the patient's outlook (eg ploidy, blastocyst karyotype, and early morphological response). In 2000, the GCC and POG teamed up to form the Children's Oncology Group (COG). This union allowed the analysis of clinical, biological, and early response data for the prediction of event-free survival (SAI) in acute lymphoblastic leukemia (ALL), the development of a new classification system and an algorithm for treatment. Among 11,779 children (aged 1 to 21.99 years) with new diagnoses of ALL precursor B, who then entered GCC (December 1988 to August 1995, n = 4,986) and POG (January The study retrospectively analyzed 6,238 patients (from GG 1.182; POG, 5.056) with informative cytogenetic data (Schultz et al., Blood 109 (2007): 926-935). Four risk groups were defined as very high risk (VHR, with 5-year EFS of 45% or less), lower risk (with 5-year EFS of at least 85%), standard and high-risk (those that remained in the respective National Cancer Institute (NCI) risk groups). The criteria for VHR included extreme hypodiploidy (less than 44 chromosomes), t (9; 22) and / or BCR / ABL, and failed induction. Patients at low risk were those in the NCI standard risk group who had both t (12; 21) (TEL / AML1) and simultaneous trisomies of chromosomes 4, 10, and 17. Even with differences in treatment, there was agreement between GCC and POG analyzes. The COG risk classification scheme is being used to divide B precursor ALL into lower (27%), standard (32%), high (37%) and very high (4%), with based on age, white blood cell count (WBC), cytogenetics, bone marrow response at day 14, and residual minimal disease (MRD) at the end of induction by flow cytometry in COG assays.

Currently, the assignment of risk-based treatment is used for pediatric or children's acute lymphoblastic leukemia (ALL). This methodology allows assigning modest therapy to children who have a history of good outcomes, avoiding more intensive and toxic treatments, while allowing children with a lower probability of long-term survival in their history to be treated with more intensive therapy than can increase your chances of healing. It was found that older children and adolescents (> 10 years) and newborns (<12 months) had less favorable prognoses than children 1 to 9 at the time of diagnosis, and were generally more aggressive treatments for these patients (Nachman J, Br. J. Haematol, 130 (2005): 166-73). Treatment with the CD19xCD3 single stranded bispecific antibody now provides a better, less toxic therapy for these populations of pediatric patients ie older children and adolescents (> 10 years) and newborns (&lt; 12 months) , with less favorable results in conventional ALL therapy, such as chemotherapy and / or HSCT. Successful treatment of children with ALL requires control of systemic disease (eg, bone marrow, liver and spleen, lymph nodes) as well as prevention or extramedullary disease, especially in the central nervous system (CNS) Only 3% of patients have detectable CNS involvement by conventional criteria at diagnosis (&gt; 5 WBC / microliter of lymphoblastic cells present). Unless specific therapy is directed at the CNS, however, 50% to 70% or more of the children will eventually develop CNS-stated leukemia. It is therefore recommended today that all children with ALL receive systemic combination therapy in conjunction with some form of CNS prophylaxis. Most sites today treat patients with CNS-diagnosed leukemia (> 5 WBC / pL with blasts; CNS3), and those with T cell phenotype and high WBC count at diagnosis, by intrathecal therapy and irradiation cranial. Thus, a treatment with the single chain bispecific antibody CD19xCD3 is preferably carried out in combination with the prophylaxis to the CNS, such as intrathecal therapy and / or cranial irradiation. Conventional or standard treatment for children with ALL is divided into stages: remission induction, consolidation or enhancement therapy, and maintenance (or continuation) therapy, and a CNS sanctuary therapy is generally provided at each stage. A phase of intensification of therapy after induction of remission is used for all patients. The intensity of both induction therapy and post-induction therapy is determined by the clinical and biological prognostic factors used for treatment based on the risk assessment and some initial response types. This evaluation may include the percentage of blasts in the marrow on day 7 and / or day 14, a peripheral blood blasts count on day 8, and determinations of minimal residual disease in the bone marrow and / or peripheral blood during or at the end of induction (Pui CH, Evans WE, N. Engl. J. Med. 354 (2006): 166-78). The duration of therapy for children with ALL ranges from 2 to 3 years. In contrast, a very rapid response to treatment with CD19xCD3 single stranded bispecific antibody in pediatric patients can be observed, as illustrated in the examples which follow. In addition, patient 1 remained negative for MRD to date (November 2009), indicating that it was possible to achieve a long-term cure.

Subgroups of patients with unfavorable prognosis with conventional or standard current therapy may require different treatment. For example, ALL newborns are at a higher risk of treatment failure, with the worst prognosis in those with MLL genetic transpositions (Rubnitz JE, et al .: Blood 84 (1994): 570-3; Biondi A , et al., Blood 96 (2000): 24-33; Pui CH, et al., Lancet 359 (2002): 1909-15, Silverman LB, et al., Cancer 80 (1997): 2285-95). These children are generally treated using regimens designed specifically for newborns (Silverman, et al., (1997), loc. Cit., Chessells JM, et al., J. Haematol, 117 (2002): 306-14; Reaman GH, et al., J. Clin Oncol 17 (1999): 445-55; Pieters R, et al., Lancet 370 (2007): 240-50). Current regimens for newborns employ intensified treatment routes and may provide better disease control as compared to earlier, less intensive methodologies, but the long term outcome and toxicity are not known (Reaman (1999), loc. Cit. Kors et al., Blood 104 (2004): 3527-34; Hilden JM, et al., Blood 108 (2006): 441-51). Some children (older than 1 year) with ALL may have a probability of long-term remission below 50% with current therapy (eg t [9; 22] Philadelphia chromosome positive ALL, hypodiploid patients, and those with failure of the initial induction). For these patients, an allogeneic bone marrow transplant of a sibling with antigens (HLA) of paired human leukocytes is considered during the first remission (Snyder DS, et al., Leukemia 13 (1999): 2053-8; Aricò M, et al. al., N. Engl. J. Med. 342 (2000): 9, 98-1006, Schrauder A, et al., J. Clin Oncol 24 (2006): 5742-9). A transplant of a paired HLA sibling, however, has not proved to be of benefit to patients defined as being at high risk only through their WBC count, their gender, and their age (Ribera JM, et al., J. Clin Oncol 25 (2007): 16-24).

Since myelosuppression and generalized immunosuppression are an early consequence of both leukemia and its treatment with chemotherapy, patients have to be closely monitored during conventional treatments of pediatric ALL. Appropriate facilities should be available immediately for both hematological support and for the treatment of complications, both infectious and others, throughout all stages of leukemia treatment. About 1% of patients die during induction therapy and more than 1% to 3% die during the first remission of treatment-related complications (Christensen MS, et al., Br. J. Haematol, 131 (2005) : 50-8). Treatment with the single chain bispecific antibody CD19xCD3 provides an alternative and less toxic therapy of pediatric ALL, especially for refractory ALL and / or relapses of pediatric ALL. The above treatment avoids the drawbacks of conventional ALL therapies in children, such as treatment failure, toxicity and long term adverse effects. It is therefore a very efficient alternative but less toxic and with less health risk, in relation to chemotherapy and / or allogeneic HSCT.

Therefore, in another embodiment of the pharmaceutical methods of the invention, said acute lymphoblastic leukemia (ALL) is refractory ALL and / or a relapse of ALL. Pediatric ALL may be refractory to chemotherapy or hematopoietic allogeneic stem cell transplantation (HSCT) or to chemotherapy and hematopoietic allogeneic stem cell transplantation (HSCT). ALL may be a relapse of ALL or a relapse of ALL refractory to conventional ALL therapy, including chemotherapy and / or hematopoietic gemstal cell (HSCT) transplantation. However, it is also within the scope of the invention that ALL is a newly diagnosed ALL. Under these conditions, treatment with the single chain bispecific antibody CD19xCD3 can be used as the first (frontline) therapy, either alone or in combination with HSCT. The term &quot; ALL refractory pediatric &quot; as used herein means pediatric ALL resistance to conventional or ALL standard therapy, such as chemotherapy and / or HSCT. Nowadays, the relapse rate in pediatric ALL is around 25%. In other words: Conventional or standard ALL therapy is ultimately not able to cure all pediatric patients. The term &quot; pediatric ALL relapse &quot; as used herein denotes the return of signs and symptoms of ALL disease after a pediatric patient has achieved remission. For example, following a conventional treatment of ALL using chemotherapy and / or HSCT, a patient with ALL may enter remission without any sign or symptom of ALL, remain in remission for a couple of years, but then relapse and than being treated again by ALL.

Pediatric patients with ALL relapses often have refractory disease. These patients are very susceptible to chemotherapy-related toxicity, which can be avoided by treatment with the single chain bispecific antibody CD19xCD3. The term &quot; standard therapy &quot; or &quot; conventional therapy &quot; as used herein refers to a pediatric ALL therapy with chemotherapy and / or HSCT. Pediatric acute lymphoblastic leukemia (ALL), including pediatric acute lymphoblastic leukemia of precursor B and other types of pediatric ALL with B cell lineage, and their treatments, have been reviewed in, for example, Pui CH, Clin. Adv. Hematol. Oncol. 4 (2006): 884-6; Pui CH, Evans WE, N. Engl. J. Med. 354 (2006): 166-178; Pui CH et al., Lancet 371 (2008): 1030-1043; Pui CH, Jeha S, Nat. See, Drug Discov. 6 (2007): 149-165); Henze G, von Stackelberg A, Relapsed acute lymphoblastic leukemia. In: Childhood Leukemias, C-H Pui editor Cambridge: Cambridge University Press; 2006, pp. 473-486). More information about pediatric ALL can also be found, for example, at http://www.cancer.gov or at http: // www. leukemia-lymphoma.org. The term &quot; single-stranded bispecific antibody &quot; or &quot; single-chain and bispecific antibody &quot; or related terms thereto according to the present invention, means constructs of antibodies resulting from joining at least two variable regions of antibodies in a single polypeptide chain free of the constant and / or Fc moieties present in the whole immunoglobulin. The single chain bispecific antibody as referred to herein is functional, i.e., cytotoxically active, as a monomer, and is therefore clearly distinguishable from the diabodies or tandabs described in the art and which are only functional as dimers or multimers. A &quot; binding agent &quot; as used herein binds the V domains with the same specificity, while a &quot; spacer agent &quot; as used herein links the V domains with different specificities. For example, a single chain bispecific antibody may be a construct having in total two antibody variable regions, for example two VH regions, each capable of specifically binding to a separate antigen, and bound to one another by an antibody (generally less than 10 amino acids), which is a synthetic polypeptide, such that the two antibody variable regions with their intervening spacer exist as a single, contiguous polypeptide chain. Another example of a single chain bispecific antibody may be a single polypeptide chain with three variable antibody regions. In this case, two antibody variable regions, for example a VH and a VL, may constitute a scFv, wherein the two variable regions of the antibody are linked to each other through a synthetic polypeptide binding agent, the latter being generally obtained by genetic engineering so as to be minimally immunogenic while maintaining maximum resistance to proteolysis. This scFv is capable of specifically binding to a specific antigen, and is linked to a further variable antibody region, for example a VH region, capable of binding to a different antigen than that to which it binds scFv. Yet another example of a single chain bispecific antibody may be a single polypeptide chain with four variable antibody regions. In this case, the first two antibody variable regions, for example a VH region and a VL region, may form a scFv capable of binding to an antigen, while the second VH region and the second VL region may form a second scFv capable of binding to an antigen. if it binds to another antigen. In a single contiguous polypeptide chain individual antibody variable regions with a specificity may be advantageously separated by a polypeptide linker as described above, the respective scFv may advantageously be separated by a short polypeptide chain from a spacer, such as described above. Non-limiting examples of bispecific single chain antibodies, as well as methods for producing them, are found in WO 99/54440, WO 2004/106381, WO 2007/068354, Mack, J. Immunol. (1997), 158, 3965-70; Mack, PNAS, (1995), 92, 7021-5; Kufer, Cancer Immunol. Immunother., (1997), 45, 193-7; Loftier, Blood, (2000), 95 (6) 2098-103; Briihl, J.

Immunol., (2001), 166, 2420-2426.

As used herein, &quot; CD3 &quot; denotes an antigen which is expressed on T cells, preferably human T cells, as part of the T cell multimolecular receptor complex, with CD3 consisting of five different chains: CD3-epsilon, CD3-gamma, CD3-delta, CD3-eta and CD3 zeta. When CD3 is accumulated in T cells, for example due to anti-CD3 antibodies, T cell activation is generated similar to an antigen binding but independent of the clonal specificity of the T cell subset. Thus, the term &quot; CD19xCD3 single chain bispecific antibody &quot; as used herein refers to a specific construct for CD3, capable of binding to the human CD3 complex expressed on human T cells and capable of inducing the deletion / lysis of target cells in that said target cells carry / display an antigen which is attached to the non-CD3 binding portion of the single chain bispecific antibody. Binding to the CD3 complex by CD3 specific binding agents (for example a single chain bispecific antibody as administered according to the pharmaceutical methods of the invention) leads to the activation of T cells as is known in the art; see, for example, WO 99/54440 or WO 2007 / 068,354. Accordingly, a construction suitable for the pharmaceutical methods of the invention is advantageously capable of eliminating / lyping target cells in vivo and / or in vitro. Corresponding target cells include cells expressing a tumor antigen, such as CD19, which is recognized by the second specificity (i.e., the non-CD3 binding portion of the single-stranded bispecific antibody) of the aforementioned construct. Preferably, said second specificity is for human CD19 that has already been described in WO 99/54440, WO 2004 / 106.381 or WO 2007 / 068,354. According to this embodiment, each antigen-specific portion of the single-chain bispecific antibody includes a VH antibody region and a VL antibody region. An advantageous variant of this single-stranded bispecific antibody is, from the N-terminus to the C-terminal: VL (CD19) -VH (CD19) -VH (CD3) -VL (CD3).

In the meaning of the invention, the term &quot; specifically binding &quot; or related terms such as &quot; specificity &quot; should be understood as being characterized primarily by two parameters: a qualitative parameter (the binding epitope, or to which an antibody binds) and a quantitative parameter (the binding affinity, or the intensity at which this antibody binds where turns on). Advantageously, the epitope to which an antibody binds can be advantageously determined using, for example, the FACS methodology, ELISA, epitope mapping by peptide spots, or mass spectroscopy. The strength with which the antibody binds to a well-defined epitope can be advantageously determined for example by using well known Biacore and / or ELISA methodologies. A combination of these techniques allows a signal to noise ratio to be calculated as a measure representative of the binding specificity, the signal represents the strength of the binding of the antibody to the epitope of interest, while the noise represents the strength of the antibody binding to other, unrelated, different epitopes of the epitope of interest. A signal to noise ratio of, for example, at least 50, but preferably about 80, for the respective epitope of interest and as determined for example by Biacore, ELISA or FACS may be taken as an indication that the evaluated antibody is binds to the epitope of interest in a specific way, i.e. it is a &quot; specific binder &quot;. The term &quot; connect to / interact with &quot; may also relate to a conformational epitope, a structural epitope or a discontinuous epitope consisting of two or even more regions of the human target molecules or their parts. A conformational epitope is defined as two or more discrete amino acid sequences separated in the primary sequence, which approach the surface of the molecule when the polypeptide folds to the native protein (Sela, (1969) Science 166, 1365 and Laver, (1990) Cell 61, 553-6). The term &quot; discontinuous epitope &quot; means non-linear epitopes which are assembled from residues from regions distant from the polypeptide chain. These residues approach the surface of the molecule when the polypeptide chain folds to a three-dimensional structure to form a conformational / structural epitope. The term &quot; treatment &quot;, as used herein, means in its broadest sense medicinal procedures or applications intended to alleviate a disease. In the present case, administration of the single chain bispecific antibody CD19xCD3 (prepared for administration to a pediatric patient with ALL), as described herein, is intended for the treatment, amelioration or elimination of ALL pediatric disease. The term &quot; improvement &quot; as used herein is synonymous with improvement. When the condition of a pediatric patient with ALL indicates improvement, the patient is clearly better - there is some improvement in their clinical condition. For example, it may be an improvement in the patient's condition with ALL, when stabilization of ALL disease is achieved, i.e. when the ALL disease no longer shows progression. This stage of disease is also called stable disease. An improvement may also be to achieve a better MRD status on the part of the pediatric patient with ALL. For example, prior to treatment with the single chain bispecific antibody CD19xCD3, 100 leukemia cells can be detected in every 104 bone marrow cells for the pediatric patient with ALL. Due to treatment with the single chain bispecific antibody CD19xCD3, the number of leukemic cells can be decreased to 10 leukemic cells or even fewer leukemic cells (for example less than one leukemic cell) in every 104 bone marrow cells in this case example . The term &quot; elimination &quot; as used herein means the removal of the leukemic cells from the body of a pediatric patient with ALL. As shown in the following example, administration of the single chain bispecific antibody CD19xCD3 is capable of transforming MRD positive (ALL) positive lymphoblastic leukemia to a negative state for MRD, i.e. a state in which there is no detectable MRD (<1CT4, i.e. less than 1 leukemia cell detectable by 104 bone marrow cells). In this case, complete molecular remission was achieved. The term &quot; administration &quot; as used herein means the administration of a therapeutically effective dose of the aforementioned single chain bispecific CD19xCD3 antibody (preferably corresponding to SEQ ID NO: 1) to a subject , i.e. a human pediatric patient. A &quot; therapeutically effective amount &quot; means a dose that produces the effects for which it is administered, i.e., sufficient to kill acute lymphoblastic leukemia cells. Preferably, the dose of the single chain bispecific antibody CD19xCD3 leads to the elimination of all acute lymphoblastic leukemia cells from the pediatric patient's body, resulting in a condition of ALL negative for MRD, as defined herein. The exact dose will depend on the purpose of the treatment, and may be identified by a person skilled in the art using known methodologies. The attending physician and clinical factors determined the dosing regimen. As is known in the medical arts, dosages for a particular pediatric patient depend on many factors, including the size of the pediatric patient, the surface area of his body, his age, his body mass, the specific compound he is about to administer , its gender, the height and route of administration, its general health, and the other drugs being administered during the same period.

A typical dose may be, for example, within the ranges described in the embodiments of the methods of the invention and the accompanying examples; however, smaller or larger doses relative to this exemplary range are also envisaged, particularly paying attention to the factors mentioned above. The term &quot; continuous infusion &quot; refers to an infusion that is allowed to continue continuously over a period of time, that is, without interruption. &quot; Infusion Continues &quot; refers to the infusion administered on an ongoing basis. Therefore, in the context of the invention, the terms &quot; permanent &quot; and &quot; continuous &quot; are used as synonyms. Within the meaning ascribed in the invention, for example the term &quot; administered by continuous infusion for (at least) 4 weeks &quot; or the like, denote a situation in which the single chain bispecific antibody CD19xCD3 used in the pharmaceutical methods according to the invention is continuously administered to the body of a pediatric patient over a period of (at least) 4 weeks in a sustained, constant , over the entire period of time required according to the pharmaceutical means and the methods of the invention. Schemes for continuous administration of the single chain bispecific antibody CD19xCD3 are described in more detail in WO 2007/068354. There is no interruption of the introduction of the single chain bispecific antibody CD19xCD3 during the total period of time that lasts for administration according to the pharmaceutical methods of the invention, with no transition occurring between a state in which this antibody is being administered to the body of the pediatric patient, and a condition in which this antibody is no longer being administered to the body of the pediatric patient, or does not occur significantly, for reasons other than re-infilling the infusion system with the single chain bispecific antibody CD19xCD3 being administered, or medicinal interventions that become necessary, and the like. As far as said filler means a temporary interruption of the introduction of the administered antibody, this administration should still be considered as &quot; uninterrupted &quot; or &quot; permanent &quot;, in the sense of pharmaceutical methods according to the invention. In most cases, since the duration of such interruption for filling will generally be short or even infinitesimal compared to the time period during which the regime of administering the antibody to the body of the pediatric patient was planned according to the pharmaceutical methods of the invention. invention. According to the invention, a course of treatment can be understood as a continuous infusion of the single chain bispecific antibody CD19xCD3 to the patient with ALL for a period of (at least) 4 weeks, followed by a treatment-free interval of 2 weeks. The term &quot; as &quot; as used herein means that the continuous infusion may also be carried out for a period of time greater than 4 weeks, for example for 5, 6, 7 or 8 weeks, or followed by a treatment-free interval of 2 weeks. It may be the case that in the determination of MRD of treated pediatric patients after a period of (at least) 4 weeks of continuous administration (or a course of treatment), a minimal or partial response is observed but not a negativity for MRD . Under these circumstances, continuous administration for a further one, two, three, four or even up to ten cycles of treatment may be extended to achieve a better therapeutic result, for example a complete haematological response, or even a complete molecular response. Preferably, said complete molecular response will be a negativity for MRD (as defined hereinbelow), which decreases the risk of ALL disease relapse. For example, negativity to MRD or low levels of MRD in pediatric patients with ALL prior to an allogeneic HSCT has been shown to decrease the risk of relapse (Bader P, et al., J. Clin. Oncol. : 377-384). As illustrated in the following examples, negativity for MRD can be achieved by treatment of pediatric patients with ALL with the single chain bispecific antibody CD19xCD3.

Therefore, in one embodiment of the pharmaceutical methods of the invention, the treatment cycle (s) during which the single chain bispecific antibody CD19xCD3 is administered continuously to the pediatric patient with ALL is or is followed by an allogeneic HSCT. In other words: In this preferred embodiment the method of the invention applies prior to allogeneic stem cell transplantation (HSCT), to transform the positive ALL for MRD to a negative condition for MRD. In this way, the risk of relapse is significantly reduced.

In another embodiment of the method of the invention, the method is used after a transplantation of allogeneic hematopoietic stem cells (HSCT). The transplantation process may be followed by one or more cycles of treatment with the single chain bispecific antibody CD19xCD3. This embodiment is important in cases of relapsed pediatric ALL in which patients have already received chemotherapy and HSCT. Due to the failure of these conventional therapies, the disease may recur and be cured by the administration of the aforementioned antibody. In addition, treatment by the single chain bispecific antibody CD19xCD3 can be used as consolidation therapy after HSCT to prevent another relapse of ALL disease.

The following examples also provide data on the use of the single chain bispecific antibody CD19xCD3 following an allogenic HSCT, wherein the mobilized T cells are derived from the donor. A powerful induction of a graft-versus-leukemia (GvL) effect in the absence of graft versus host disease (GvHD) induced by treatment with the single-chain bispecific antibody CD19xCD3 was observed in two patients with chemorhiferative relapse of an ALL CD19 + after an allogeneic hematopoietic HSCT. Therefore, in another preferred embodiment of the pharmaceutical methods of the invention, the pediatric patient with ALL can be treated with the single chain bispecific antibody CD19xCD3 after receiving an allogeneic HSCT.

In a best case scenario, it is envisaged that the treatment of pediatric patients with ALL using the single chain bispecific antibody CD19xCD3 would be able to replace conventional pediatric ALL therapy such as chemotherapy and / or allogeneic hematopoietic stem cell transplantation (HSCT ).

As shown in the following examples, the treatment of patients with acute pediatric lymphoblastic leukemia (ALL) with the single chain bispecific antibody CD19xCD3 is capable of eliminating acute lymphoblastic leukemia cells from patients' bodies to levels below the threshold of detection. Preferably, the main therapeutic objective of administration of the single chain bispecific antibody CD16xCD3, either alone or in combination with an allogeneic HSCT, to a pediatric patient with ALL, is the transformation from a positive condition for MRD to a negative condition for MRD , resulting in leukemia-free survival, as defined hereinbelow. As demonstrated in this document, pediatric patients with ALL positive for MRD became negative for MRD after the first course of treatment in which the single chain bispecific antibody CD19xCD3 was continuously administered for five weeks (patient 1) or for four weeks (patient 2). In patient 1, the course of treatment was followed by a haploid HSCT. Until November 2009, this patient remains in complete remission negative for MRD, ie, tumor free.

Continuing the uninterrupted administration of the single chain bispecific antibody CD19xCD3 according to the pharmaceutical methods of the invention for longer periods of time allows the advantageous T cell activation mentioned in the examples to exert its effect for a time sufficient to advantageously eliminate all acute lymphoblastic leukemia cells from the body. Since the rate of administration of the single chain bispecific antibody administered continuously is kept small, the application of the therapeutic agent may be continued for a longer time without the risk of harmful side effects to the patient. The single chain bispecific antibody CD19xCD3 as used herein is advantageously prepared as a pharmaceutical composition for administration to a human pediatric patient diagnosed with acute lymphoblastic leukemia (ALL). Said ALL may be a ALL diagnosed at the time, an ALL refractory to chemotherapy and / or allogeneic haematopoietic stem cell transplantation (HSCT), a relapse of ALL or a relapse of an ALL refractory to chemotherapy and / or cell transplantation allogeneic hematopoietic stem cells (HSCT).

In a preferred embodiment of the pharmaceutical methods of the invention, said pediatric acute lymphoblastic leukemia (ALL) is pediatric acute lymphoblastic leukemia B (ALL), preferably a pediatric acute lymphoblastic leukemia precursor B. The vast majority of cases of pediatric ALL > 8,5%) are cells of the precursor B phenotype. Several classifications of ALL with B lineage have been proposed (see, for example, Schultz et al., Blood 109 (2007): 926-935). In order to modulate the intensity of therapy relative to the risk of relapse of a patient, patients with precursor B ALL are now stratified into &quot; small &quot; standard &quot; &quot; &quot; large &quot; or &quot; too large &quot; using laboratory and clinical parameters: patient's age, gender, white blood cell count (WBC) when the disease presents, and the presence or absence of specific cytogenetic abnormalities. Cytogenetic abnormalities that occur frequently and help define these risk groups include, for example: t (12; 21) [TEL-AML1]; t (1; 19;) [E2A-PBX]; t (4; 11) [AF4-MLL]; t (9; 22) [BCR-ABL]; hyperdiploidy (or trisomy of chromosomes 4, 10, and 17), and hypodiploidy; see for example Schultz et al. ; Bader et al., Loc. cit. Using data from several studies, a classification system was developed and implemented, such as COG AALL03B1 (Classification of Acute Lymphoblastic Leukemia) (Raetz et al., Personalized Med 2 (2005), 349-361; Schultz et al. , Blood 109 (2007): 926-935). Since the single chain CD19xCD3 bispecific antibody described herein is directed against the marker associated with B cells, CD19, this antibody is especially suitable as a therapeutic agent for pediatric lymphoblastic leukemia with lineage B, preferably for precursor B's ALL. Pediatric precursor B ALLs can be further subdivided into pediatric ALL pro-B, ALL pre-B, and ALL common (cALL). As shown in the following examples, treatment according to the methods of the invention of the seven-year old patient with common ALL refractory against any other and therefore probably fatal treatment resulted not only in complete haematological remission but also in molecular remission complete. In other words, it was no longer possible to detect any minimal residual disease in this patient after treatment with the single chain bispecific antibody CD19xCD3. Particularly preferably, pediatric acute lymphoblastic leukemia (ALL) is a precursor B ALL, more preferably cALL. Importantly, the single chain bispecific antibody CD19xCD3 is capable of killing not only ALL cells with TCR or immunoglobulin transpositions, but also ALL cells with several other cytogenetic abnormalities: For example, there was found in patient 2 described in the following examples as well as in adult ALL patients the said antibody is capable of treating an ALL characterized by immunoglobulin or TCR transpositions, t (4; ll) translocations or bcr / abl (Ph +) fusion transcripts. In particular, ALL Ph + and ALL with t (4; ll) translocations had been described as being extremely difficult to treat by standard ALL therapy but could be treated successfully with the single chain bispecific antibody CD19xCD3. The mentioned data indicate that the single chain bispecific antibody CD19xCD3 is capable of treating various forms of ALL including for example ALLs characterized by t (12; 21) [TEL-AML1]; t (1; 19;) [E2A-PBX]; t (4; 11) [AF4-MLL]; t (9; 22) [BCR-ABL]; hyperdiploidy (or trisomy of chromosomes 4, 10, and 17), hypodiploidy as well as immunoglobulin or TCR transpositions; see also Table 1. The diagnosis of pediatric ALL is based on morphological, cytochemical and immunological characteristics of cells, including the morphology of lymphoblasts in Wright-Giemsa contrast-enhanced plating of bone marrow, positive contrast for terminal deoxynucleotide transferase (TdT) , negative contrast for myeloperoxidase, and expression on the cell surface of 2 or more lymphoid differentiation antigens for precursor B cells. Immunological phenotyping has been described, for example, by Behm FG (Immunophenotyping, Childhood Leukemias, C-H Pui editor, Cambridge: Cambridge University Press, 2006, pp. 150-209), and can be carried out for example by indirect immunofluorescence, immunohistochemistry and / or flow cytometry. The term &quot; patient &quot; as used herein refers to a human patient. The term &quot; ALL pediatric &quot; or &quot; pediatric ALL patient &quot;, as referred to herein, denotes young people between one month and 18 years of age. The indicated age should be understood as the age of the young when diagnosing any of ALL diseases. Young people may be more particularly grouped into subgroups of newborns (1 to 12 months of age), younger children aged 1 to 9 years, and younger children and adolescents (> 10 to 18 years of age). As used herein, a time interval which is defined as &quot; X to Y &quot; means the same as a time interval defined as &quot; between X and Y &quot;. Both of these time intervals include in particular the upper limit and also the lower limit. This means for example that a time interval of &quot; between one month and 18 years &quot; includes both &quot; one month &quot; as &quot; 18 &quot;. Preferably, the patient to be treated according to the present invention will be at most 18 years of age (including patients aged 18 years).

As shown in the examples which follow, patient 1 was seven years old when treated with the single chain bispecific antibody CD19xCD3, ALL was diagnosed at two years of age. Patient 2 was fifteen years old when treated with the single chain bispecific antibody CD19xCD3, ALL was diagnosed in 2001.

The above definitions apply mutatis mutandis to the term &quot; ALL acute pediatric lymphoblastic leukemia (ALL) &quot; &quot; ALL in children &quot;, or to similar ones. For example, ALL or pediatric ALL should be understood as the ALL diagnosed in a pediatric patient between 1 month (including 1 month) and 18 years (including 18 years) of age.

Although the single chain bispecific antibody CD19xCD3, as used herein, may be administered by itself, administration to a pharmaceutical carrier is preferred. Examples of suitable pharmaceutical carriers are well known in the art and include solutions in phosphate buffered saline, water, liposomes, with various types of wetting agents, sterile solutions, etc. Compositions comprising these carriers can be formulated using well-known conventional methods. These pharmaceutical compositions may be administered to the pediatric patient at a suitable dose. The dosing regimen will be determined by the attending physician taking into account the clinical factors. As is well known in medicine, dosages for any pediatric patient depend on many factors, including the patient's size, surface area of the body, age, body mass, specific compound to be administered, its gender, the height and route of administration, its general health, and the other drugs being administered to it at the same time. Aqueous or non-aqueous sterile solutions, or suspensions, are included in the preparations for parenteral administration. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, and injectable organic esters such as ethyl oleate. Water carriers, aqueous solutions, or suspensions, including saline and buffered media, are included in the aqueous vehicles. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, or lactated Ringer's. Intravenous vehicles include fluid and nutrient refills, electrolyte refills (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like may also be present. In addition, the composition may include protein carriers, such as for example serum albumin or immunoglobulin, preferably of human origin. It is envisaged that the composition may contain, in addition to the single chain bispecific protein antibody, other biologically active agents depending on the intended use of the pharmaceutical composition. These agents may be agents that act as cytostatics, hyperuriquemic agents, immunological reactions inhibitors (eg, corticosteroids, FK506), drugs that act on the circulatory system and / or agents such as T-cell costimulatory molecules or cytokines known in the art. For example, treatment with the single chain bispecific antibody CD19xCD3 in combination with intrathecal chemotherapy such as for prophylaxis of the CNS, corticoids, and / or allopurinol may be carried out.

Preferably, the single chain bispecific antibody CD19xCD3 as defined herein is formulated into a buffer, with a stabilizer and a surfactant. The buffer may be a phosphate, citrate, succinate or acetate buffer. The stabilizer may be one or more amino acids) and / or a sugar. The surfactants may be detergents, PEG, or the like. More preferably, the single chain bispecific antibody CD19xCD3 as defined herein is formulated into citrate, lysine, trehalose and Tween 80. As the diluent for the pharmaceutical composition of the invention, isotonic saline and Tween 80 are preferred.

Preferably, in the pharmaceutical methods of the invention, the pharmaceutical composition is prepared for administration to a human pediatric patient to whom acute lymphoblastic leukemia (ALL) has been diagnosed. The success of single chain CD19xCD3 bispecific antibody therapy can be monitored by standard methods well established for the respective entities in the disease: For B-cell ALL therapy, Fluorescence Activated Cell Distinction (FACS) can be used, bone marrow aspiration and various clinical chemical parameters specific for leukemia, as well as other standard methods established and known in the art. Methods and means for determining minimal residual disease (MRD) status are described herein.

The cytotoxic activity of CD19xCD3 single stranded bispecific antibody can be determined by methods known in the art and methods such as those illustrated for example in WO 99/54440, WO 2004/106381, WO 2007/068354.

In a preferred embodiment of the pharmaceutical methods of the invention, the precursor ALL of the pediatric patient (s) is relapsed and / or refractory to the treatment. With current pediatric ALL treatment, the event-free survival rate is around 75%. Therefore, 25% of patients relapse despite the toxic treatment and with health risks. The problems in managing ALL relapses are the resistance of leukemic cells and the lower tolerances of pediatric patients to a second episode of treatment after they have already received intensive therapy of the front line, resulting in a lower remission rate as well as a higher incidence of subsequent relapses and a lower overall result. Intensified polychemotherapy is then essential to induce a second complete remission (Henze G, von Stackelberg A, Relapsed acute lymphoblastic leukemia, Childhood Leukemias, C-H Pui publisher, Cambridge: Cambridge University Press, 2006, pp. 473-486). The term &quot; refractory to chemotherapy and / or allogeneic stem cell transplantation &quot; as used herein means that pediatric patients with ALL are resistant to these therapies and therefore relapse after a standard ALL treatment. Pediatric patients who relapse after treatment with the CD19xCD3 single chain bispecific antibody are also envisioned to receive one or more cycles of treatment with said antibody to render the children treated MRD negative. Said patients can then for example receive a second allogeneic HSCT.

As shown in the examples which follow, the pharmaceutical methods of the invention are especially suitable for the treatment of pediatric patients who are resistant to conventional ALL therapies. Although pediatric patients in these examples have received strong prior treatments with both chemotherapy and allogeneic stem cell transplantation, patients have relapsed several times. The patients therefore had an extremely unfavorable prognosis. However, after treatment with the single chain bispecific antibody CD19xCD3, patients were negative for MRD, i.e., denoted complete molecular remission. In other words: The treatment eliminated acute lymphoblastic leukemia cells from the body of pediatric patients up to less than the limit of detection. In a way that is most important, the bone marrow of the pediatric patients was cleansed of leukemic cells.

In a preferred embodiment of the pharmaceutical methods of the invention, acute lymphoblastic leukemia (ALL) of the pediatric patient (s) is refractory to chemotherapy and / or allogeneic HSCT with respect to MRD. In other words: MRD in pediatric patients with ALL is resistant to chemotherapy and / or allogeneic HSCT.

It is contemplated that the pharmaceutical methods of the invention will also be suitable for the treatment of newly diagnosed pediatric ALL as it provides an alternative to conventional pediatric ALL regimens such as allogeneic chemotherapy and / or HSCT.

In yet another preferred embodiment, said acute lymphoblastic leukemia (ALL) falls within three years of diagnosis, preferably within two years of diagnosis, even more preferably in the year following diagnosis. Preferably, said relapse is a relapse in the bone marrow. The term &quot; chemotherapy &quot; as used herein denotes chemotherapy used for the treatment of pediatric acute lymphoblastic leukemia (ALL). Chemotherapy is the preferred initial treatment for pediatric ALL (see, for example, Pui CH, Eja S, Nat. See Drug Drug, 6 (2007): 149-165; Schmiegelow K, Gustaffson G. Acute lymphoblastic Leukemia. : Cancer in Children: Clinical Management, Voute PA,

Barret A, Stevens MCG, Caron HN (eds.), London, UK, Oxford University Press, 2005, pp. 138-170; Schmoll, Hoffken, Possinger, loc. cit.). Most pediatric patients with ALL end up receiving a combination of different treatments. In the treatment of pediatric ALL, there are no surgical options due to the distribution of malignant cells throughout the body. In general, cytotoxic chemotherapy for pediatric ALL combines multiple anti-leukemic drugs in several combinations. Chemotherapy for pediatric ALL consists of three phases: induction of remission, intensification, and maintenance therapy. Chemotherapy is also indicated to protect the central nervous system from leukemia. The goal of induction of remission is to rapidly kill most of the tumor cells and to take the pediatric patient to complete hematologic remission. This is defined as a presence of less than 5% of leukemic blasts in the bone marrow (as determined by light microscopy). For example, they are used to induce remission, alone or in combination, Clofarabine, Cyclophosphamide, VP16, Amsacrine, Prednisone, Melphalan, or Cytarabine. Intensification uses high intravenous doses of multiple drug chemotherapy to decrease tumor burden. The typical protocols of enhancement are vincristine, cyclophosphamide, cytarabine, daunoorubicin, etoposide, thioguanine or mercaptopurine given as blocks in different combinations. Since ALL cells sometimes penetrate the Central Nervous System (CNS), most protocols include the delivery of chemotherapy to the CNS fluid, which is usually referred to as intrathecal chemotherapy. Some cancer centers administer the drug via an Ommaya reservoir (a device placed surgically under the scalp and used to deliver drugs to the CNS fluid and to extract fluid from the CNS for various tests). Other centers perform a number of lumbar perforations as needed for testing and medication delivery. In general, methotrexate or cytarabine is used intrathecally for this purpose. The goal of maintenance therapy is to kill any residual leukemic cells that have not been killed during the remission induction and enhancement regimens. Although these cells are few, they will relapse if they are not eliminated. For this purpose, daily mercaptopurine, weekly oral methotrexate, a monthly cycle of 5 days of intravenous vincristine are used. The duration of maintenance therapy is 3 years for boys, 2 years for girls and for adults. It involves relapse in the nervous system by intrathecal administration of hydrocortisone, methotrexate, and cytarabine. Since chemotherapy regimens can be intensive and prolonged (often about 2 years in the GMALL UKALL, HyperCVAD or CALGB protocols, about 3 years for males under COG protocols), many patients are undergoing an intravenous catheter inserted into a large vein (called a central venous catheter or a Hickman line), or a Portacath (a cone-shaped delivery device with a silicone lump that is surgically implanted under the skin, usually near a collarbone). However, chemotherapy continues to be a highly toxic procedure, especially for pediatric patients.

Patients may experience relapse of ALL following initial therapy and / or become refractory to chemotherapy following treatment. Patients with pediatric ALL who are refractory to chemotherapy have markedly unfavorable prognoses. In particular, the prognosis for pediatric patients with ALL Ph + or ALL with t (4; 11) translocations treated with chemotherapy alone is unfavorable. Since the method of the invention is capable of rendering pediatric ALL patients negative for MRD, it is especially useful for the treatment of ALL patients refractory to chemotherapy and / or allogeneic HSCT. Under these conditions, the term &quot; refractory to chemotherapy and / or allogeneic HSCT &quot; as used herein denotes resistance of acute lymphoblastic leukemia cells to chemotherapy and / or allogeneic HSCT. The term &quot; allogeneic hematopoietic stem cell transplantation (HSCT) &quot; as used herein means hematopoietic stem cell transplantation (HSCT) or bone marrow transplantation which is a medical procedure in the field of hematology and oncology which involves the transplantation of hematopoietic stem cells (HSC). It is most often carried out in patients with blood or bone marrow diseases, or suffering from certain types of cancer, such as ALL. Most recipients of HSCT are patients with leukemia (for example ALLs that would benefit from treatment with high doses of chemotherapy or with whole body irradiation. Allogeneic HSCT in children with ALL has been described, for example, in Schrauder A, et al. However, allogeneic stem cell transplantation remains a risky procedure, especially for pediatric patients. The term &quot; eligible for allogeneic haematopoietic stem cell transplantation (&quot; HSCT) &quot; as used herein means that allogeneic stem cell transplantation is the necessary therapy for the pediatric ALL patient. In cases in which the pediatric ALL patient is eligible for allogeneic stem cell transplantation, In the first, in one embodiment of the pharmaceutical methods of the invention administration of the single chain bispecific antibody CD16xCD3 (alone but preferably in the form of a pharmaceutical composition) may be used to replace allogeneic stem cell transplantation which is used as a standard therapy for patients with eligible pediatric ALL for transplantation. Therefore the method of the invention can avoid the health risks of all pediatric ALL patients associated with allogeneic HSCT. In addition, about 30% of patients with transplanted pediatric ALL relapse after transplantation. Therefore the method of the invention can be used to treat these patients. In the alternative embodiment, continuous infusion of the single chain bispecific antibody CD19xCD3 in the patient with pediatric ALL may be followed by allogeneic stem cell transplantation. In this embodiment, administration of a pharmaceutical composition comprising a single chain bispecific antibody construct CD19xCD3 can be used to transform pediatric ALL patients eligible for transplantation to a negative state for MRD before they are given the transplantation. Thus, the method of the invention can be used to eliminate MRD, which leads to a lower risk of relapse than in the transplantation treatment of MRD positive patients. The following examples present a pediatric patient (patient 1) that was first transformed to a negative condition for MRD by treatment with the single chain bispecific antibody CD19xCD3, followed by allogeneic transplantation. To date (November 2009), this pediatric patient is still negative for MRD. The term &quot; ineligible for allogeneic hematopoietic stem cell (HSCT) transplantation &quot;, as used herein, means those pediatric patients for whom allogeneic stem cell transplantation is not the ALL recommended treatment, for example , due to medical reasons. It could also happen that no suitable donor was available for the transplantation of allogeneic stem cells.

Thus, in one embodiment, acute lymphoblastic leukemia (ALL) is refractory to chemotherapy and / or allogeneic HSCT in pediatric patients not eligible or no longer eligible for an allogeneic HSCT. For example, a pediatric patient may be in such a poor state of health that no transplantation of allogeneic stem cells can be carried out for medical reasons. Patient 1 shown in the following examples was not eligible for allogeneic stem cell transplantation prior to treatment with the single chain bispecific antibody CD19xCD3 due to its poor health. In such a case, treatment with the single chain bispecific antibody CD19xCD3 provides a novel route of treatment for pediatric ALL.

Until now, ALL meant a death sentence for pediatric patients refractory to chemotherapy and not eligible for allogeneic HSCT. The method of the invention provides for the first time a therapy for this population of pediatric patients in that it eliminates the minimal residual disease (MRD) that would otherwise cause relapse and ultimately kill the referred patients.

It is within the scope of the pharmaceutical methods of the invention to administer the single chain bispecific antibody construct CD19xCD3 to pediatric ALL patients who previously received chemotherapy, alone or in combination with allogeneic stem cell transplantation, and then fell.

In another preferred embodiment, the method of the invention is for the treatment, amelioration or elimination of minimal residual disease (MRD) in a pediatric patient with acute lymphoblastic leukemia (ALL). The term &quot; minimal residual disease (MRD) &quot; as defined herein denotes a term that is used after a treatment, for example with chemotherapeutics, when no longer can find leukemic cells in the bone marrow by the standard tests , such as by microscopy. Instead, more sensitive tests such as flow cytometry (FACS-based methods) or polymerase chain reaction (PCR) have to be used to find evidence that leukemia cells have remained in the bone marrow of the patient with ALL pediatric population. More in particular, the presence of leukemic cells in less than the cytological detection limit (5% of leukemic cells) is defined as minimal residual disease (MRD). When no MRD is detected (<1CT4, i.e. detecting less than 1 leukemia cell in every 104 bone marrow cells), complete molecular remission was achieved (negativity for MRD or negative for MRD) . A &quot; MRD positive state &quot; as defined herein means a signal measured by PCR or by FACS above its detection limit or quantitative threshold. A &quot; MRD negative status &quot; as defined herein means that it is below the detection limit and / or below the quantitative threshold measured by PCR or by FACS. The value of the classification in terms of quantification of minimal residual disease in children's ALL has been described, for example, in Bader et al. (J. Clin Oncol. 27 (2009): 377-384) or in Eckert et al. (Lancet 358 (2001): 1239-41). The condition for MRD can be measured by PCR or FACS analysis, as the individual cytogenetic abnormalities mentioned herein, and / or transpositions of immunoglobulin or T cell receptor (TCR) genes, are quantitatively detected. For example, a PCR analysis can detect fusion transcripts such as bcr / abl or t (4; ll) translocations as well as individual clonal transpositions of immunoglobulins (IgH) and / or T cell receptor (TCR) genes.

As demonstrated in the following examples, after treatment with the single chain CD19xCD3 bispecific antibody described herein, all cells of acute lymphoblastic leukemia could be eliminated from the bodies of patients with pediatric ALL, thereby achieving a complete molecular remission (i.e., a negative condition for MRD).

Recurrent chromosomal abnormalities in malignant cells in pediatric and adult patients suffering from acute lymphoblastic leukemia are the universal signs of this disease (Harrison and Foroni, Rev. Clin. Exp. Hematol 6 (2002), 91-113). Particular aberrations that are often indicative of underlying molecular lesions are consistent and can assist or even establish the diagnosis and determine optimal therapy. In many children, many good, high-risk cytogenetic subgroups have been identified, which are commonly used to stratify patients by recommending specific therapies (Pui and Evans, N. Engl. J. Med. 354 (2006), 166-178) . In adult ALL, the role of cytogenetics in patient management has largely been centered on the presence of the Philadelphia chromosome (Ph) that usually comes from t (9; 22) (q34; qll.2) and gives rise to BCR-ABL fusion Faderl et al., Blood 91 (1998), 3995-4019). Although the overall incidence of ALL Ph + ALL in adults is about 25%, it correlates with age and increases to more than 50% in patients over 55 years of age (Appelbaum, American Education Book 2005 Society of Clinical Oncology, Alexandria: ASCO, 2005: 528-532). Other cytogenetic translocations associated with specific genetic molecular abnormalities in acute lymphoblastic leukemia (ALL) are listed in Table 1 and have also been described in Schultz et al. or in Bader et al., loc. cit.

Cytogenetics has been increasingly recognized as an important tool in predicting the outcome of ALL (Moormann et al., Blood 109 (2007), 3189-97).

Prognostics for some cytogenetic subtypes are worse than for others. These include for example: (i) A translocation between chromosomes 9 and 22, the Philadelphia chromosome (Ph +) occurs in about 20% of ALL cases in adults and 5% in pediatric patients. (ii) A translocation between chromosomes 4 and 11 occurs in about 4% of ALL cases and is more common in neonates with less than months.

Transpositions of immunoglobulin genes or T cell receptor (TCR) transpositions and their role in ALL have been described in the art (see, for example, Szczepahski et al., Leukemia 12 (1998), 1081-1088).

In another preferred embodiment of the pharmaceutical methods of the invention, said patient with pediatric ALL is positive for MRD but in complete haematological remission. The term &quot; forgiveness &quot; or &quot; complete haematological remission &quot; as used herein should be understood to mean that there are no signs of ALL disease following a standard treatment, for example after chemotherapy and / or transplantation. This means that the bone marrow contains less than 5% of blast cells as determined by light microscopy, that blood cell counts are within normal limits, and that there are no signs or symptoms of ALL disease. However, it may happen that not all leukemic cells have been eliminated from the body. One of these patients, although classified as being in remission or complete hematologic remission, is still positive for MRD. These remaining tumor cells can cause a relapse of leukemia. The pharmaceutical methods of the invention can be used to kill these remaining tumor cells to prevent relapse of leukemia due to hidden leukemic cells remaining in the body after primary therapy. Thus, pharmaceutical media and methods help prevent relapse of the disease in all pediatric ALL patients.

In contrast, a &quot; complete molecular remission &quot; means that there are no traces of leukemic cells in bone marrow biopsies, even when very sensitive tests such as PCR or FACS analysis are used. In other words: When no MRD (&lt; 1CT4, i.e., less than one leukemia cell per 104 bone marrow cells) is detectable, complete molecular remission was achieved.

In another preferred embodiment of the pharmaceutical methods of the invention, administration of said pharmaceutical composition transforms MRD-positive pediatric acute lymphoblastic leukemia (ALL) to a condition that is negative for MRD.

In another preferred embodiment of the pharmaceutical methods of the invention, the MRD is measured by the quantitative detection of at least one of the cytogenetic abnormalities or transpositions, selected from the group consisting of: TEL-AML1; t (1; 19;) [E2A-PBX]; t (4; 11) [AF4-MLL]; t (9; 22) [BCR-ABL]; hyperdiploidy or simultaneous trisomies on chromosomes 4, 10, and 17; hypodiploidy (i.e., less than 44 chromosomes); transpositions of immunoglobulin genes; and transpositions into the T cell receptor genes.

MRD is measured with quantitative detection of at least one of: (i) the individual cytogenetic abnormalities referred to herein, such as t (12; 21) (TEL-AML1); t (1; 19;) [E2A (BCR-ABL), hyperdiploidy (or simultaneous trisomies on chromosomes 4, 10, and 17), hypodiploidy (i.e., less than or (ii) transpositions of the immunoglobulin genes or into T cell receptors (TCRs), see also Table 1. Said quantitative detection of the mentioned cytogenetic abnormalities or transpositions is preferably carried out using, The method of the invention provides a therapeutic route for the treatment, amelioration or elimination of MRD, thereby decreasing or even abolishing the risk of relapse for the patient with pediatric ALL. no curative treatment of MRD has been available to date in patients with ALL pedi attrition.

In another preferred embodiment of the pharmaceutical methods of the invention, said pediatric patient with an MRD denotes a signal having a cytogenetic abnormality above the detection limit and / or at least one transposable label with a sensitivity of> 10 -4. Preferably, the MRD is detected by analysis using PCR and / or FACS.

Individual cytogenetic abnormalities mentioned herein include for example t (12; 21) [TEL-AML1]; t (1; 19;) [E2A-PBX]; t (4; 11) [AF4-MLL]; t (9; 22) [BCR-ABL]; hyperdiploidy (or trisomy of chromosomes 4, 10, and 17), hypodiploidy. Transpositions include, for example, transpositions of the immunoglobulin or T cell receptor (TCR) genes; see also Table 1. The MRD represented by ALL cells bearing said cytogenetic abnormalities and / or transpositions can be detected by a PCR or FACS analysis.

For example, the term &quot; bcr / abl signal above the detection limit &quot; as used herein means that a PCR or FACS analysis leads to a detectable bcr / abl signal. Similarly, a signal of a translocation t (4; ll) means that said translocation can be detected by PCR or by FACS. This applies mutatis mutandis to the other cytogenetic abnormalities or to the transpositions mentioned herein. The term &quot; with a sensitivity &gt; 1CT4 &quot; in the context of the transpositions, means that at least one leukemic cell or more (with a TCR or immunoglobulin transposition mentioned) per 104 cells of bone marrow can be detected by the methods mentioned.

In another preferred embodiment of the pharmaceutical methods of the invention, the time to molecular relapse (detectable by the assays described above) is longer than 6 months, preferably more than 7, 8, 9, 10, 11 or 12 months, or still more preferably 2, 3, 4, 5 or more years. The term &quot; molecular relapse &quot; as used herein means that said pediatric patient has a signal of a cytogenetic abnormality above the detection limit and / or at least one transposable marker with a sensitivity of &lt; 10 4 , by PCR and / or by FACS, as set forth above.

In another preferred embodiment of the pharmaceutical methods of the invention, the corresponding heavy chain (VH) variable regions and the corresponding light chain (VL) variable regions in the construction of said single chain bispecific antibody CD19xCD3 are arranged, from the N-terminus to terminal C, in the order VL (CD19) -VH (CD19) -VH (CD3) -VL (CD3).

The corresponding heavy chain (VH) variable regions and the corresponding light chain (VL) variable regions of the CD3 binding domains and the CD19 of the single chain bispecific antibody CD19xCD3 respectively in SEQ ID Nos. 3 to 10. The corresponding CDR regions of the respective VH and VL regions of said single chain bispecific antibody CD19xCD3 are shown by SEQ ID Nos. 11 to 22.

In another preferred embodiment of the pharmaceutical methods of the invention, the construction of said single chain bispecific antibody CD19xCD3 includes an amino acid sequence as set forth in SEQ ID NO. 1, or an amino acid sequence that is at least 90%, preferably at least 95% identical to SEQ ID NO. 1. The invention describes a single chain bispecific antibody molecule CD19xCD3 which includes an amino acid sequence as set forth in SEQ ID NO. 1, as well as an amino acid sequence that is at least 90% or preferably 95%, and preferably at least 96, 97, 98, or 99% identical to the amino acid sequence of SEQ ID NO. 1. The invention also describes the corresponding nucleic acid sequence as in SEQ ID NO. 2, as an identical nucleic acid sequence at least 90%, preferably 95% identical, and preferably at least 96, 97, 98, or 99% identical to the nucleic acid sequence represented by SEQ ID NO. 2. It is to be understood that sequence identity is determined over the entire nucleotide or amino acid sequence. Furthermore, it is to be understood that a single-chain bispecific antibody molecule which includes an amino acid sequence that is at least 90% or preferably 95% identical, and preferably 96, 97, 98, or 99% identical to the sequence of amino acids of SEQ ID NO. 1 contains all of the CDR sequences shown in SEQ ID Nos. For the sequence alignments, for example, the Gap or BestFit programs (Needleman and Wunsch J. Mol. Biol. 48 (1970), 443-453; Smith and Waterman, Adv. Appl. Math 2 (1981), 482-489) contained in the GCG (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin 53711 (1991) program package. , nucleotide or amino acid sequences having, for example, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the nucleotide or amino acid sequences of the single chain bispecific antibody CD19xCD3 described For example, and according to Crick's Wobble hypothesis, the 5 'base of the anti-codon does not have the same stereochemical limitations as the other two bases, and may therefore have a base pairing different from that of In other words: the third position of the triplet of a codon may be varied such that two triplets differing in this third position may encode the same amino acid residue. This hypothesis is well known to those skilled in the art (see for example http://en.wikipedia.org/wiki/Wobble_Hypothesis; Crick, J. Mol. Biol. 19 (1966): 548-55). Further it is a routine procedure for those skilled in the art to determine the cytotoxic activity of such a nucleotide or amino acid sequence having an identity of, for example, 90%, 95%, 96%, 97%, 98%, or 99% , with the nucleotide or amino acid sequence of the single chain bispecific antibody CD19xCD3 described herein. The cytotoxic activity of the single chain bispecific antibody CD19xCD3 or an antibody construct having, for example, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the amino acid sequence of bispecific antibody CD19xCD3 can be determined by methods such as those shown, for example, in WO 99/54440, WO 2004/0103681, or WO 2007 / 068.354.

In another preferred embodiment of the pharmaceutical methods of the invention, the pharmaceutical composition containing a single chain bispecific antibody construct CD19xCD3 is to be administered by continuous infusion for at least four weeks followed by a 2 week interval without any treatment. A course of treatment is understood as a continuous infusion of said antibody for at least four weeks, followed by a 2 week interval without any treatment. This range may in turn be followed by one or more cycles of treatment or by an allogeneic HSCT. Preferably, said administration by continuous infusion of CD19xCD3 single chain bispecific antibody (i.e. one treatment cycle) should be repeated at least two, three, four, five, six, seven, eight, nine or even ten times, then of a negative state for MRD, for consolidation.

In one embodiment of the pharmaceutical methods of the invention, the method is used prior to transplantation of allogeneic stem cells to transform the MRD-positive state of ALL into the negative for MRD.

In another embodiment of the pharmaceutical methods of the invention, the methods are employed after an allogeneic HSCT, for example, in cases where pediatric ALL has been relapsed following conventional ALL therapy using chemotherapy and / or allogeneic stem cell transplantation.

In another preferred embodiment of the pharmaceutical methods of the invention, the construction of the single chain bispecific antibody CD19xCD3 is administered at a daily dose of between 10 pg and 100 pg per square meter of the patient's body surface area.

As used herein, a dosage range defined as &quot; X to Y &quot; means a dose range defined as &quot; between X and Y &quot; . This range includes the upper limit and also the lower limit. This means, for example, that a daily dose of 10 pg to 100 pg per square meter of body surface area includes &quot; 10 pg &quot; and &quot; 100 pg &quot; .

In an even more preferred embodiment of the pharmaceutical methods of the invention, the administration of the single chain bispecific antibody CD19xCD3 at a daily dose of 15æg, 30æg, 60æg or 90æg per square meter of body surface area is administered. It is still more preferred to administer the antibody at a daily dose of between 15 and 30 pg per square meter of the body surface area of the patient, preferably at a daily dose of 15 or 30 pg per square meter of the surface area of the patient. body of the patient. The mean body surface area of a pediatric patient is calculated herein and in the context of the method or use according to the invention as being between about 0.2 and 2.2 m 2. For this calculation see for example the formulas provided at http: //www.cato .eu / koerperoberflaeche-kinder.html

In another embodiment of the pharmaceutical methods of the invention, pediatric acute lymphoblastic leukemia (ALL) has just been diagnosed, is ALL refractory or a relapsed and pediatric, or is a refractory ALL relapse. A pediatric ALL just diagnosed, as used herein, means that the ALL disease was first diagnosed in the pediatric patient.

In another embodiment of the pharmaceutical methods of the invention, said method is directed to a patient at high risk of relapse depending on the COGAALL03B1 classification of acute lymphoblastic leukemia.

Advantageously, the pharmaceutical composition containing the single chain bispecific antibody CD19xCD3 as recited herein also optionally comprises (a) one or more reaction buffers, storage solutions and / or additional agents or materials necessary for the method or use described. In addition, said components may be individually packaged in bottles or bottles, or in combination in containers or units with various containers.

In order to assess the safety and tolerability of CD19xCD3 single chain bispecific antibody as described herein, the compound is administered by a continuous long-term infusion.

It has been found that the beneficial and unexpected effects of the pharmaceutical methods of the invention can be obtained by administering the single chain bispecific antibody CD19xCD3 at a daily dose of between 10 micrograms and 100 micrograms per square meter of body surface area. The constant dose may be maintained throughout the administration period. However, it is also within the scope of this embodiment that on the or the early days of the infusion period, a smaller dose of the single chain bispecific antibody CD19xCD3 (&quot; initial dose &quot;) may be administered prior to that disclosed in the pharmaceutical methods described herein document, while a larger dose (&quot; maintenance dose &quot;) is applied during the remaining period. This procedure can assist in the adaptation of the patient's body to treatment with the antibody and / or avoid undesirable side effects. For example, 5 micrograms of single chain bispecific antibody per square meter of body surface area may be administered as the initial dose on or on the first days (for example on the first day, or on the first and second days, or on the three first days, etc. up to the seventh day) of the infusion period, followed by administration of 15 micrograms per square meter body surface area as a daily dose over the remaining period. Or 5 micrograms of the single chain bispecific antibody per square meter of body surface area may be administered as a starting dose on or in the first days of the infusion period, followed by administration of 30 or 45 micrograms per square meter of body surface area as a daily dose over the remaining period. Five micrograms of CD19xCD3 single chain bispecific antibody per square meter of body surface area during the first few days of the infusion period is also administered, followed by administration of 15 micrograms of the single chain bispecific CD19xCD3 antibody by square meter of body surface area during the following days of the infusion period, thereafter 30 or 45 micrograms per square meter of body surface area as a daily (maintenance) dose is administered for the remainder of the infusion period, for a total of at least 4 weeks. The two initial doses may be administered not only for one day, but also for 2, 3, 4, 5, 6 or 7 days or longer. To this end, the mean surface area of a patient's body in the context of the pharmaceutical methods according to the invention is between 0.2 and 2.2 m 2. Uninterrupted administration of CD19xCD3 single chain bispecific antibody may be intravenous, parenteral, subcutaneous, transdermal, intraperitoneal, intramuscular or pulmonary. The mode of intravenous administration will be the method chosen in most cases for the uninterrupted administration of the single chain bispecific antibody CD19xCD3 and, where appropriate, for the co-administration of a pharmaceutical agent incorporating a co-therapy regimen. Thus, intravenous administration is especially preferred. In this case, a suitable metering device such as a multi-therapy infusion pump, for example a model 6060 pump manufactured by Baxter, may be advantageously selected. Whichever measuring device is selected, it must be of such a design and construction that it is minimized or even better that an interruption of the administration of the therapeutic agent is avoided when exchanging cartridges and / or batteries or energy cells, or their recharging. This can be achieved, for example by selecting a device with a secondary reservoir of the CD19xCD3 single chain bispecific antibody solution, in addition to the cartridge to be replaced, so as to ensure a continuous infusion from this secondary reservoir and to the patient even when removing the empty or almost empty cartridge and replacing it with a new one.

One mode of intravenous and, where appropriate, co-administration as part of a co-therapy regimen involves implantation of a pump into the body of the pediatric patient to quantify the referred administration. These metering pumps are known to those skilled in the art, for example the 6060 model manufactured by Baxter as mentioned above. By way of non-limiting example, it may be the case that uninterrupted, i.e. continuous, administration is carried out by a small pump system that may be affixed or implanted in the pediatric patient to measure the influx of the therapeutic agent into the body of the patient. These pump systems are generally known in the art, and are generally based on the periodic exchange of fillers containing the therapeutic agent to be infused. When the reservoir of such a pump system is exchanged, a temporary interruption of the otherwise uninterrupted flow of the therapeutic agent into the body of the pediatric patient may occur. In such a case, the pre-charge phase administration phase and the delivery phase immediately after the charge replacement would still be considered, within the scope of the pharmaceutical methods of the invention, as constituting a component of &quot; uninterrupted administration &quot; of said therapeutic agent. The same would apply to very long-lasting administrations in which the load would have to be replaced more than once, or in the case where the batteries or batteries that pump the pump need replacement, leading to a temporary stop of the therapeutic solution flow into the patient's body.

Appropriate measures should also be taken to minimize the danger of infection at the point where it perforates to administer the drug to the patient's body, as these long-lasting wounds are especially prone to infections. This also applies to intramuscular administration using a similar delivery system. Continued administration may be transdermal via a patch affixed to the skin and replaced at intervals. One skilled in the art is aware of systems with drug delivery pads that are suitable for this purpose. It should be noted that transdermal administration is particularly usable for uninterrupted administration since replacement of a first depleted patch can advantageously be made upon placement of a new patch, for example on the surface of the skin immediately adjacent to that of the first and immediately prior to the removal of this first depleted patch. The problems of flow interruption or power failure are not set.

Further, the invention relates to a single-chain bispecific antibody construct CD19xCD3 for use in a method of treating, ameliorating or eliminating pediatric acute lymphoblastic leukemia (ALL), wherein said single-chain bispecific antibody CD19xCD3 is prepared for administration to a pediatric patient. The invention also relates to the use of a CD19xCD3 single stranded bispecific antibody construct for the preparation of a pharmaceutical composition for the treatment, amelioration or elimination of pediatric acute lymphoblastic leukemia (ALL). Therefore, the present invention also relates to a single-chain bispecific CD19xCD3 antibody construct as defined herein for the treatment, amelioration or elimination of pediatric acute lymphoblastic leukemia (ALL). The embodiments described herein in the context of pharmaceutical methods will apply mutatis mutandis to the preparation of the corresponding pharmaceutical composition including anti-CD19xCD3 single chain constructs for administration to a pediatric patient, in particular in the treatment, amelioration or elimination of acute lymphoblastic leukemia ( ALL) pediatric.

Preferably, said pediatric acute lymphoblastic leukemia (ALL) is pediatric acute lymphoblastic leukemia (ALL) of B-lineage, preferably a pediatric B-precursor acute ALL (ALL), most preferably pediatric ALL, pre-B ALL pediatric or pediatric ALL (cALL). In the most preferred case of all, ALL is cALL.

In a preferred embodiment of the aforementioned medicinal uses, said precursor B ALL is a relapse of ALL and / or an ALL refractory to conventional treatment for ALL, such as chemotherapy and / or HSCT.

Preferably, said acute lymphoblastic leukemia (ALL) falls within the first three years after its diagnosis.

In another preferred embodiment of the aforementioned medicinal uses, the construction of the single chain bispecific antibody CD19xCD3 is for the treatment, amelioration or elimination of minimal residual disease (MRD) in a pediatric patient with acute lymphoblastic leukemia (ALL). Preferably, the said pediatric patient is MRD positive in complete haematological remission.

In a further preferred embodiment of the aforementioned medicinal uses, administration of said single chain bispecific antibody CD19xCD3 transforms pediatric acute lymphoblastic leukemia (ALL) from a positive condition for MRD to a negative condition for MRD.

Preferably, MRD is measured by quantitative detection of individual cytogenetic abnormalities or transpositions, as defined herein, using PCR and / or FACS analysis. Even more preferred, the patient with pediatric ALL denotes a signal of a cytogenetic abnormality above the detection limit and / or at least one transposable marker with a sensitivity of &lt; 10-4.

In another preferred embodiment of the aforementioned medicinal uses, the time to molecular relapse detectable by the indicated detection methods is longer than 6 months.

In another preferred embodiment of the aforementioned medicinal uses, the corresponding heavy chain (VH) variable regions and the corresponding light chain (VL) variable regions in said single chain bispecific antibody construct CD19xCD3 are disposed, from the N-terminus and up to terminal C, in the order VL (CD19) -VH (CD19) -VH (CD3) -VL (CD3). Preferably, the construction of said single chain bispecific antibody CD19xCD3 includes an amino acid sequence as described in SEQ ID NO. 1, or an amino acid sequence that is at least 90%, preferably 95%, identical to SEQ ID NO. 1.

In another preferred embodiment of the aforementioned medicinal uses, a course of treatment is a continuous infusion for at least four weeks, followed by a treatment-free 2-week interval.

Preferably, said administration will be repeated two, three, four, five, six, seven, eight, nine or ten times, after a negative condition for MRD has been determined.

In another embodiment, the administration is done prior to a transplantation with allogeneic stem cells to transform an ALL into a positive condition for MRD, in a condition negative for MRD.

In an alternate embodiment, administration occurs following a transplantation with allogeneic stem cells.

In another preferred embodiment of the preferred medical uses, the construction of the single chain bispecific antibody CD19xCD3 will be administered at a daily dose of between 10 pg and 100 pg per square meter of body surface area of the patient.

Preferably, the CD19xCD3 single chain bispecific antibody construct is administered at a daily dose of 15æg to 30æg per square meter of body surface area of the patient.

In another embodiment of preferred medicinal uses, administration of the CD19xCD3 single chain bispecific antibody construct replaces allogeneic stem cell transplantation in pediatric patients eligible for allogeneic stem cell transplantation.

The embodiments, definitions and explanations provided with respect to the pharmaceutical methods of the invention apply mutatis mutandis to the medicinal uses of the CD19xCD3 single chain bispecific antibody described herein.

The Figures show:

Figure 1: Mode of action of the single chain bispecific antibody CD19xCD3 (CD19xCD3 bscab). The single chain bispecific antibody CD19xCD3 (SEQ ID NO.1) re-directs CD3 positive cytotoxic cells to eliminate CD19 antigen-bearing acute lymphoblastic leukemia cells.

Figure 2: Failure of conventional ALL therapy in a 7-year-old patient suffering from common ALL. One year after HSCT, the pediatric patient had a second bone marrow relapse and received subsequent chemotherapy including 1 cycle Clofarabine / Cyclophosphamide / VP16, 2 cycles of Amsacrine, VP16, prednisone and 1 cycle of Melphalan / Cytarabine. Despite this aggressive chemotherapy, the patient had a persistent morphologic disease throughout the treatment, as demonstrated by the high levels of MRD. The left yy axis indicates the MRD values in% as determined by FACS, the right yy axis indicates the MRD values in% determined by PCR (from 10 ° to <1CT4), the xx axis indicates the days from of the first HSCT; see Example 4.

Figure 3: Successful treatment of the pediatric patient by administration of the single chain bispecific antibody CD19xCD3 (CD19xCD3 bscab; SEQ ID NO: 1), as demonstrated by MRD before, during and after treatment with the above antibody. After failure of conventional therapy for ALL, the patient was treated with a continuous infusion of 15 pg / m 2 of single chain bispecific antibody CD19xCD3 (SEQ ID NO: 1) for 5 weeks. Upon treatment with the antibody, a negative condition for MRD was achieved. In October 2008, that is 2 weeks after completion of treatment with the single chain bispecific antibody CD19xCD3 (day 0 in Figures 3 to 5), a second HSCT was applied to the patient from its haploident mother using a regimen of non-myeloablative preparation consisting of Clofarabine, Thiotepa and melphalan (Lang) indicated by the wrapping bar. The left yy axis indicates the MRD values in% as determined by FACS, the right yy axis indicates the MRD values in% determined by PCR (from 10 ° to <1CT4), the xx axis indicates the days from of the first HSCT; the day &quot; 0 &quot; indicates the day of the second HSCT (after the antibody treatment); see Example 4.

Figure 4: Development over time of the blast counts before and after treatment with the single chain bispecific antibody CD19xCD3 (CD19xCD3 bscab; SEQ ID NO 1). A bone marrow analysis at day 10 of treatment with the single chain bispecific antibody CD19xCD3 revealed complete elimination of bone marrow blasts (MRD &lt; 1CT 4) by the antibody. Another BM analysis at the end of treatment with the single chain bispecific antibody CD19xCD3 at day 35 confirmed the complete absence of leukemic blasts in the bone marrow (MRD &lt; 1CT4). The left yy axis indicates the blasts counts by flow cytometry, in%, the right yy axis indicates the blasts count by microscopy, in%, the xx axis indicates days from the HSCT, indicating the negative days days relative to the first HSCT, indicating the day &quot; 0 &quot; the day of the second HSCT (after treatment with the antibody) and indicating the positive days the days after the second HSCT.

Figure 5: Decrease in MRD values by treatment with the single chain bispecific antibody CD19xCD3 (CD19xCD3 bscab; SEQ ID NO.1). Upon treatment with the antibody, a negative condition for MRD could be achieved. As of June 2009, the patient remains in a negative state for MRD in complete molecular remission. The left yy axis indicates the% MRD by FACS, the right yy axis indicates the MRD by PCR (from 10 ° to <10 -4), the xx axis indicates days from the HSCT, indicating the negative days the days from the first HSCT and indicating &quot; 0 &quot; the day of the second HSCT and the positive days indicating the days after the second HSCT. The invention is further illustrated by the following examples:

Examples: 1. Single chain bispecific antibody CD16xCD3 The generation, expression and cytotoxic activity of the single chain bispecific antibody CD19xCD3 has been described in WO 99/44440. The corresponding amino acid and nucleic acid sequences are shown for the

CD19xCD3 single chain bispecific antibody in SEQ ID Nos. 1 and 2. The VH and VL regions of the CD3 binding domain of the single chain bispecific antibody CD19xCD3 are respectively depicted in SEQ ID Nos. 7 to 10, while the VH and VL regions of the CD19 binding domain of the single chain bispecific antibody CD19xCD3 are respectively depicted in SEQ ID Nos. 3 to 6. 2. Phenotypic lymphocyte analysis and chimerism analysis

For phenotypic analysis of lymphocytes and for chimerism analysis, blood samples were obtained from the patient before, during and after treatment with the single chain bispecific antibody CD19xCD3 in sample collection tubes containing EDTA. Absolute numbers of lymphocytes in blood samples were determined by differential blood analysis on an Advia. Lymphocytes were contrasted using fluorescence labeled antibodies directed against CD3, CD4, CD8, CD19 and CD56 (all obtained from Becton-Dickinson, Heidelberg, Germany). The labeled cells and the data collection were performed on a FACSCalibur (Becton-Dickinson).

3. MRD detection

For the detection of MRD, either a polymerase chain reaction (PCR) assay (Bader et al., Loc. Cit.) Or a FACS analysis was used. In summary, the DNA was isolated with a DNeasy kit (Blood & Tissue Kit) (Qiagen GmbH, Hilden, Germany). Transpositions of immunoglobulin and T-cell receptor genes as targets based on PCR have recently been shown to be stable markers for monitoring MRD in acute lymphoblastic leukemia after stem cell transplantation (Kreyenberg, Leukemia, 2009). 4. Case reports 4.1 Patient case report 1 This common 7-year old patient was diagnosed with a common high-risk CD10 + ALL (positive for CD19-, CD34-, CD45-reduced, TCR-transposition, negative for the CNS ) in 2004. After treatment, he relapsed in the bone marrow in June 2006 and was treated according to the ALL-REZ BFM study in his S3 branch. After two cycles of chemotherapy, the patient had persistent disease and achieved complete remission after 3 treatments with clofarabine. In 2007, an allogeneic HSCT was made from a donor unrelated to 9 of 10 paired HLA alleles after whole body irradiation and with Etoposide. One year after the HSCT, he relapsed in the bone marrow and was given subsequent chemotherapy including 1 cycle of

Clofarabine / Cyclophosphamide / VP16 (Nobuko), 2 cycles of Amsacrine, VP16, prednisone (Hamburg) and 1 cycle of Melphalan / Cytarabine; see Figure 2. In spite of this aggressive chemotherapy, the patient had persistent morphological disease throughout the treatment as evidenced by the high MRD values in Figure 2. He was then treated by compassion, with a continuous infusion 15 pg / m2 of the single chain bispecific antibody CD19xCD3 (blinatumomab; SEQ ID NO. 1) for 5 weeks; see Figure 3. Serial analysis of lymphocyte populations before (day 0) and during treatment with the above-mentioned antibody denoted an impressive expansion of CD8 + T lymphocytes, with no change in CD4 + T cells and cells NK CD56 +. With the treatment using the antibody a negative condition for MRD was reached. A concomitant analysis of chimerism revealed 100% chimerism of the donor. As shown in Figure 4, a bone marrow analysis at day 102 of treatment with the single chain bispecific antibody CD19xCD3 revealed complete elimination of bone marrow blasts (MRD <1CT4). Another bone marrow analysis at the end of treatment with the single chain bispecific antibody CD19xCD3 again denoted a complete absence of leukemic blasts in the bone marrow (MRD <1CU4). The MRD assay series is represented before, during and after treatment with the antibody referred to in Figure 5. Before treatment with the antibody, the pediatric patient had a very unfavorable prognosis and was not eligible for an HSCT because of its poor clinical status. Upon treatment with the antibody, a negative condition for MRD was achieved. In addition to transient and mild signs of ataxia, no significant side effects were observed, and no signs of GvHD were seen despite the impressive expansion of donor-derived CD8 + T lymphocytes. In October 2008, 2 weeks after the end of treatment with the single chain bispecific antibody CD19xCD3 (i.e. on day 0 of Figures 3 to 5), a new HSCT was made from its haploident mother using a non-myeloablative preparative regimen consisting of Clofarabine, Tiotepa and Melphalan (Lang). In November 2009, the patient maintained her complete remission being negative for MRD. 4.2 Report of the case of the patient 2

This 15-year-old male was diagnosed with a precursor ALL with a Philadelphia chromosome and positive for precursor CD19 B in April 2001. After chemotherapy, he received a HSCT from an identical HLA sibling in October 2001, after of a 12 Gy whole body irradiation and etoposide. In 2002, relapse was diagnosed in the bone marrow and re-relapse was achieved with matinib and chemotherapy. He then received a second HSCT from an unrelated donor in HLA in October 2004. In March 2008, a second relapse was diagnosed and treated with low dose chemotherapy and Dasatinib due to resistance to Imatinib.

After further chemotherapy with Clofarabine and Cytosine / Arabinoside, he achieved molecular remission and received a third allogeneic HSCT from his haploident father with HLA mismatch in 3 of 6 alleles and one treatment after transplantation with Dasatinib. Due to gastrointestinal hemorrhage and dilatative cardiomyopathies, Dasatinib treatment was discontinued 5 months after transplantation. In April 2009, a combined central nervous system (CNS) relapse was diagnosed with 7x109 / L blasts in the CNS and 3% of blasts in the bone marrow. The patient was then treated with Nilotinib, intrathecal chemotherapy and fractional CNS irradiation with 18 Gy. Three months after this treatment, the patient's bone marrow remained positive for MRD at a level of 1.1 xlCT3 while the CNS was free of blasts. An analysis of the peripheral blood chimerism revealed fully derived donor hematopoiesis from its haploidenteric parent.

The patient was then treated with single agent blinatumomab at 15 pg / m 2 / day for 4 weeks by continuous infusion, without any side effects. A bone marrow aspiration at the end of treatment showed complete remission without detectable MRD in the marrow, <1xlCh4. Like patient 1, patient 2 did not report any signs of GvHD during or after blinatumomab treatment. Two weeks after the end of treatment with blinatumomab, the patient experienced a transient hemolytic reaction triggered by an infection. Patient 2 is currently relapse, four weeks after treatment. He's good and has gone to school. 4.3 Summary

Drugs available to treat ALL have a small specificity and affect several other cells, causing severe side effects such as immunosuppression, bone marrow aplasia, mucositis, neuropathy, cardiotoxicity, and alopecia. New, whitish pathways with less side effects are therefore urgently needed. In addition, in some patients, ALL clones are developed with complete resistance to conventional chemotherapy, especially clones appearing after HSCT, such that drugs having a different mode of action are desirable. The efficacy of antibody-dependent toxicity through NK cell and granulocyte Fc receptors, in combination with chemotherapy, could be demonstrated for pediatric ALL using the chimeric monoclonal antibody rituximab. Currently, blinatumomab is the only antibody in clinical trials that allows the mobilization of cytotoxic T cells targeting CD19 in non-Hodgkin B-cell lymphomas and lymphatic leukemias. T cells are considered to have a potential higher cytotoxicity than the immunological cells that are mobilized by the conventional monoclonal antibodies.

It is assumed that the allogeneic GvL effect is one of the main reasons for the anti-leukemic efficacy of HSCT. Unfortunately, the occurrence of GvL is often associated with GvHD, which is still a major cause of morbidity and mortality after an allogeneic HSCT. Therefore, induction of GvL in the absence of GvHD is an intensive research topic. One pathway for the induction of a GvL effect is the infusion of donor lymphocytes (DLI). Although DLI is very effective in treating CML, it is less effective in the treatment after transplantation of ALL relapses and often leads to the occurrence of chronic GvHD with a significant impairment of the quality of life of the affected children.

One possible pathway for inducing GvL without GvHD is in vivo activation of donor-derived T lymphocytes following HSCT using small doses of the blinatumomab T-cell mobilizing antibody which is able to direct T lymphocytes against CD19-positive ALL blasts of the patient. This antibody showed impressive anti-lymphoblastic and antileukemic activity in the autologous situation but was never tested in ALL relapses after HSCT nor in children. In the autologous situation, molecular remissions were induced in 13 of 16 adult patients evaluable with ALL positive for MRD. Both pediatric patients described above showed an impressive anti-leukemic response after initiation of treatment. In both cases, the MRD dropped to less than the detectable content without any signs of GvHD, despite a brutal expansion of donor-derived T cells. This may be due to the fact that the action of blinatumomab is independent of regular presentation of peptide antigen, and may not involve mobilization of the foolish T cell repertoire.

Dose levels of the blinatumomab as small as 15 pg / m 2 per day are sufficient to induce an expansion of the donor-derived T lymphocytes and to eliminate ALL blasts to levels below those detected in the MRD. This underlines that the mobilization of the T cells very different from the mode of action of the conventional monoclonal antibodies, that much larger doses are required and that they do not mobilize cytotoxic T cells because they do not have Fc receptors. Blinatumomab was well tolerated in both patients causing only fatigue, mild ataxia and a CTCAE grade 1-2 tremor. In patient 2, no side effects were seen despite the intensive prior treatments to which he had been subjected. Continuous intravenous infusion over several weeks was well accepted by both pediatric patients.

From this first clinical experience with pediatric ALL, the inventors conclude that blinatumomab is well tolerated and can promptly induce complete and negative hematological remissions for MRD in children with refractory disease with respect to therapy following multiple relapses of ALL with CD positive positive precursor B19 an allogeneic HSCT. It is noteworthy that none of the patients showed any signs of GvHD despite an expansion of the T lymphocytes from the donor, even in the case of a transplantation from a haploident with mismatches.

These first results indicate that the treatment of patients with pediatric acute lymphoblastic leukemia (ALL) by CD19xCD3 single stranded bispecific antibody is able to completely eliminate acute lymphoblastic leukemia cells from the bodies of pediatric patients. Importantly, this treatment resulted not only in complete hematological remission but also in complete molecular remission, as an acute lymphoblastic leukemia (ALL) positive residual disease (MRD) was transformed to a condition negative for MRD. Treatment was well tolerated. Under these conditions, administration of the CD19xCD3 single chain bispecific antibody described herein provides a better treatment option for pediatric acute lymphoblastic leukemia (ALL), especially for an ALL that is refractory to chemotherapy and / or allogeneic HSCT, and / or a relapse of ALL.

These results can be summarized in a few lines, as follows: • Two compassionate pediatric patients were treated. • Negative current status for MRD in referred patients. • Absence of significant adverse effects (AE). • All EC patients were transient without interruption of treatment.

SEQUENCE LISTING

&lt; 110 &gt; Micromet AG &lt; 120 &gt; New treatment of pediatric acute lymphoblastic leukemia &lt; 130 &gt; R2201 PCT S3 &lt; 150 &gt; 61 / 112,323 &lt; 151 &gt; 2008-11-07 &lt; 150 &gt; 61 / 221,269 &lt; 151 &gt; 2009-06-29 &lt; 150 &gt; 61 / 183,291 &lt; 151 &gt; 2009-06-02 &lt; 160 &gt; 22 &lt; 170 &gt; Patent in version 3.3

&lt; 210 &gt; 1 &lt; 211 &gt; 498 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Description of Artificial Sequence: Single-chain bispecific antibody CD19xCD3 &quot; &lt; 4 0 0 &gt; 1

Asp lie Gin Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 15 10 15

Gin Arg Ala Thr He Ser Cys Lys Ala Ser Gin Ser Val Asp Tyr Asp 20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gin Gin lie Pro Gly Gin Pro Pro 35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn He His 65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gin Gin Ser Thr 85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Gly 100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gin Val 115 120 125

Gin Leu Gin Gin Ser Gly Wing Glu Leu Val Arg Pro Gly Ser Ser Val 130 135 140

Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met 145 150 155 160

Asn Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile Gly Gin 165 170 175 Ile Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly 180 185 190

Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Thr Ala Tyr Met Gin 195 200 205

Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg 210 215 220

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr Trp 225 230 235 240

Gly Gin Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp 245 250 255 lie Lys Leu Gin Gin Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser 260 265 270

Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe, Thr Arg Tyr Thr 275 280 285

Met His Trp Val Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp lie Gly 290 295 300

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gin Lys Phe Lys 305 310 315 320

Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Thr Ala Tyr Met 325 330 335

Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala 340 345 350

Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gin Gly Thr 355 360 365

Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly 370 375 380

Ser Gly Gly Ser Gly Gly. Val Asp Asp lie Gin Leu Thr Gin Ser Pro 385 390 395 400

Ala-Ile-Met-Al-Ser-Pro-Gly-Glu-Lys-Val-Thr-Met-Thr-Cys-Arg-405 410 415

Ala Ser Ser Ser Ser Ser Ser Tyr Met Asn Trp Tyr Gin Gin Lys Ser Gly 420 425 430

Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala Ser Gly 435 440 445

Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu 450 455 460

Thr He Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gin 465 470 475 480

Gin Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu 485 490 495

Leu Lys

&lt; 210 &gt; 2 &lt; 211 &gt; 1.494 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: Single-chain bispecific antibody CD19xCD3 &quot; &lt; 400 &gt; 2 gatatccagc tgacccagtc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca aggccagcca aagtgttgat tatgatggtg atagttattt gaactggtac 120 caacagattc caggacagcc acccaaactc cteatctatg atgcatccaa tctagtttct 180 gggatcccac ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga ggatccgtgg 300 acgttcggtg gagggaccaa gctcgagatc aaaggtggtg gtggttctgg cggcggcggc 360 tccggtggtg gtggttctca ggtgcagctg cagcagtctg gggctgagct ggtgaggcct 420 gggtcctcag tgaagatttc ctgcaaggct tctggctatg cattcagtag ctactggatg 480 aactgggtga agcagaggcc tggacagggt cttgagtgga ttggacagat ttggcctgga 540 gatggtgata ctaactacaa tggaaagttc aagggtaaag ccactctgac tgcagacgaa 600 tcctccagca cagcctacat gcaactcagc agcctagcat ctgaggactc tgcggtctat 660 ttctgtgcaa gacgggagac tacgacggta ggccgttatt actatgctat ggactactgg 720 ggccaaggga ccacggtcac cgtctcctcc ggaggtggtg gatccgatat caaactgcag 780 cagtcagggg ctgaactggc aagacctggg gcctcagtga agatgtcctg caagacttct 840 ggctacacct ttactagg rt cacgatgcac tgggtaaaac agaggcctgg acagggtctg 900 gaatggattg gatacattaa tcctagccgt ggttatacta attacaatca gaagttcaag 960 gacaaggcca cattgactac agacaaatcc tccagcacag cctacatgca actgagcagc 1020 ctgacatctg aggactctgc agtctattac tgtgcaagat attatgatga tcattactgc 1080 cttgactact ggggccaagg caccactctc acagtctcct cagtcgaagg tggaagtgga 1140 ggttctggtg gaagtggagg ttcaggtgga gtcgacgaca ttcagotgac ccagtctcca 1200 gcaatcatgt crgcatctcc aggggagaag gtcaccatga cctgcagagc cagttcaagt 1260 gtaagttaca tgaactggta ccagcagaag tcaggcacct cccccaaaag atggatttat 1320 gacacatcca aagtggcttc 'tggagtccct tatcgcttca gtggcagtgg gtctgggacc 1380 tcatactctc tcacaatcag cagcatggag gctgaagatg ctgccactta ttactgccaa 1440 cagtggagta gtaacccgct cacgttcggt gctgggacca agctggagct gaaa 1494

&lt; 210 &gt; 3 &lt; 211 &gt; 124 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source

&lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 VH &quot; &lt; 400 &gt; 3

Trp Met Asn Trp Go Lys Gin Arg Pro Gly Gin Gly Leu Glu Trp Ile 35 40 45

Gly Gin lie Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Glu Ser Ser Thr Ala Tyr 65 70 75 80

Met Gin Leu Ser Ser Leu Ala Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95

Ala Arg Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp 100 105 110

Tyr Trp Gly Gin Gly Thr Thr Go Thr Will Be Ser 115 120

&lt; 210 &gt; 4 &lt; 211 &gt; 372 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source

&lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 VH &quot; &lt; 400 &gt; 4 caggtgcagc tgcagcagtc tggggctgag ctggtgaggc ctgggtcctc agtgaagatt 60 tcctgcaagg cttctggcta tgcattcagt agctactgga tgaactgggt gaagcagagg 120 cctggacagg gtcttgagtg gattggacag atttggcctg gagatggtga tactaactac 180 aatggaaagt tcaagggtaa agccactctg actgcagacg aatcctccag cacagcctac 240 atgcaactca gcagcctagc atctgaggac tctgcggtct atttctgtgc aagacgggag 300 actacgacgg taggccgtta ttactatgct atggactact ggggccaagg gaccacggtc 360 cc accgtctcct 372

&lt; 210 &gt; 5 &lt; 211 &gt; 111 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source

&lt; 223 &gt; / note = &quot; Artificial Sequence Description: Anti-CD19 VL &quot; &lt; 400 &gt; 5

Asp lie Gin Leu Thr Gin Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 15 10 15

Gin Arg Ala Thr Ile Ser Cys Lys Ala Ser Gin Ser Val Asp Tyr Asp 20 25 30

Gly Asp Ser Tyr Leu Asn Trp Tyr Gin Gin lie Pro Gly Gin Pro Pro 35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Val Ser Gly Ile Pro 50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn lie His 65 70 75 80

Pro Val Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gin Gin Ser Thr 85 90 95

Glu Asp Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110

&lt; 210 &gt; 6 &lt; 211 &gt; 333 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source

&lt; 223 &gt; / note = &quot; Artificial Sequence Description: Anti-CD19 VL &quot; &lt; 400 &gt; 6 gatatccagc tgacccagtc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atctcctgca aggccagcca aagtgttgat tatgatggtg atagttattt gaactggtac 120 caacagattc caggacagcc acccaaactc ctcatctatg atgcatccaa tctagtttct 180 gggatcccac ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggaga aggtggatgc tgcaacctat cactgtcagc aaagtactga ggatccgtgg 300 acgttcggtg gagggaccaa gctcgagatc aaa 333 &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt;

&lt; 210 &gt; 7 &lt; 211 &gt; 119 &lt; 212 &gt; PRT &lt; 221 &gt; source &lt; 223 &gt; VH anti CD3 &lt; 400 &gt; 7

Asp lie Lys Leu Gin Gin Be Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr-Ser Gly Tyr Thr Phe Thr Arg Tyr 20 25 30

Thr Met His Trp Val Lys Gin Arg Pro Gly Gin'Gly Leu Glu Trp lie 35 40 45

Gly Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gin Lys Phe 50 55 60

Lys Asp Lys Ala Thr Leu Thr Thr Asp Lys Ser Ser Thr Ala Tyr 65 70 75 80

Met Gin Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95

Ala Arg Tyr Tyr 'Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gin Gly 100 105 110

Thr Thr Leu Thr Val Ser Ser 115

&lt; 210 &gt; 8 &lt; 211 &gt; 357 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source

&lt; 223 &gt; / note = &quot; Artificial Sequence Description: Anti-CD3 VH &quot; &lt; 400 &gt; 8 gatatcaaac tgcagcagtc aggggctgaa ctggcaagac ctggggcctc agtgaagatg 60 tcctgcaaga cttctggcta cacctttact aggtacacga tgcactgggt aaaacagagg 120 cctggacagg gtctggaatg gattggatac attaatccta gccgtggtta tactaattac 180 aatcagaagt tcaaggacaa ggccacattg actacagaca aatcctccag cacagcctac 240 atgoaactga gcàgcctgac atctgaggac tctgcagtct attactgtgc aagatattat 300 gatgatcatt actgccttga ctactggggc caaggcacca ctctcacagt 357 ctcctca

&lt; 210 &gt; 9 &lt; 211 &gt; 106 &lt; 212 &gt; PRT &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 221 &gt; source

&lt; 223 &gt; / note = &quot; Artificial Sequence Description: Anti-CD3 VL &quot; &lt; 400 &gt; 9

Asp-Ile Gin-Leu-Thr-Gin-Pro-Al-Ile-Met-Ser-Ser-Pro-Pro-Gly 15 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Met 20 25 30

Asn Trp Tyr Gin Gin Lys Ser Gly Thr Ser Pro Lys Arg Trp lie Tyr 35 40 45

Asp Thr Ser Lys Val Ala Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser 50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu 65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gin Gin Trp Ser Ser Asn Pro Leu Thr 85 90 95

Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105

&lt; 210 &gt; 10 &lt; 211 &gt; 318 &lt; 212 &gt; DNA &lt; 213 &gt; Artificial Sequence &lt; 22 0 &gt; &lt; 22 &gt; &lt; 221 &gt; source &lt; 4 0 0 &gt; 10 gacattcagc tgacccagtc tccagcaatc atgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gagccagttc aagtgtaagt tacatgaact ggtaccagca gaagtcaggc 120 acctccccca aaagatggat ttatgacaca tccaaagtgg cttctggagt cccttatcgc 180 ttcagtggca gtgggtctgg gacctcatac tctctcacaa tcagcagcat ggaggctgaa 240 gatgctgcca cttattactg ccaacagtgg agtagtaacc cgctcacgtt cggtgctggg 300 accaagctgg agctgaaa 318 &lt; 213 &gt; artificial sequence &lt; 220 &gt;

&lt; 210 &gt; 11 &lt; 211 &gt; 15 &lt; 212 &gt; PRT &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 LI &quot; &lt; 4 0 0 &gt; 11

Lys Ala Ser Gin Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu Asn 15. 10 15

&lt; 210 &gt; 12 &lt; 211 &gt; 7 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 L2 &quot; &lt; 4 0 0 &gt; 12

Asp Ala Ser Asn Leu Val Ser 15 &lt; 210 &gt; 13 &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 211 &gt; 9

&lt; 212 &gt; PRT &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 L3 &quot; &lt; 4 0 0 &gt; 13

Gin Gin Ser Thr Glu Asp Pro Trp Thr 1 5

&lt; 210 &gt; 14 &lt; 211 &gt; 5 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 HI &quot; &lt; 4 0 0 &gt; 14

Ser Tyr Trp Met Asn 1 &lt; 210 &gt; 15 &lt; 211 &gt; 17 &lt; 213 &gt; artificial sequence &lt; 220 &gt;

&lt; 212 &gt; PRT &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 H2 &quot; &lt; 4 0 0 &gt; 15

Gin lie Trp Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys 1 5 10 15

Gly

&lt; 210 &gt; 16 &lt; 211 &gt; 15 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD19 H3 &quot; &lt; 4 0 0 &gt; 16

Arg Glu Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Ala Met Asp Tyr 15 10 &lt; 210 &gt; 17 &lt; 211 &gt; 5

&lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD3 HI &quot; &lt; 4 0 0 &gt; 17

Arg Tyr Thr Met His 1 5

&lt; 210 &gt; 18 &lt; 211 &gt; 17 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD3 H2 &quot; &lt; 4 0 0 &gt; 18

Tyr Ile Asn Pro Ser Arg Gly Tyr Thr Asn Tyr Asn Gin Lys Phe Lys 15 10 15

Asp &lt; 210 &gt; 19 &lt; 211 &gt; 10

&lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD 3 H3 &quot; &lt; 4 0 0 &gt; 19

Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr 1 5 10

&lt; 210 &gt; 20 &lt; 211 &gt; 10 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD3 LI &quot; &lt; 400 &gt; 20

Arg Ala Ser Ser Ser Ser Ser Ser Tyr Met Asn 15 &lt; 210 &gt; 21 &lt; 211 &gt; 7 &lt; 213 &gt; artificial sequence &lt; 220 &gt;

&lt; 212 &gt; PRT &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD3 L2 &quot; &lt; 400 &gt; 21

Asp Thr Ser Lys Val Ala Ser 15

&lt; 210 &gt; 22 &lt; 211 &gt; 9 &lt; 212 &gt; PRT &lt; 213 &gt; artificial sequence &lt; 220 &gt; &lt; 221 &gt; source &lt; 223 &gt; / note = &quot; Artificial sequence description: anti-CD 3 L3 &quot; &lt; 400 &gt; 22

Gin Gin Trp Ser Ser Asn Pro Leu Thr 1 5

Lisbon, March 18, 2015.

Claims (19)

A CD19xCD3 single stranded bispecific antibody construct for use in a method for the treatment, amelioration or elimination of pediatric acute lymphoblastic leukemia (ALL) in a pediatric ALL patient. The construct for the use of claim 1, wherein said pediatric acute lymphoblastic leukemia (ALL) is pediatric acute lymphoblastic leukemia (ALL) of B-line, preferably an acute lymphoblastic leukemia (ALL) of precursor B, more preferably ALL pro-B pediatric, a pre-B pediatric ALL or a common ALL (cALL). The construct for the use of claim 1 or 2, wherein said acute lymphoblastic leukemia (ALL) is ALL refractory and / or a relapse of ALL. The construct for the use of any one of claims 1 to 3, wherein the method is for the treatment, amelioration or elimination of minimal residual disease (MRD) in a patient with pediatric ALL. The construct for the use of claim 4, wherein said patient with pediatric ALL is positive for MRD in complete haematological remission. The construct for the use of claim 4 or 5, wherein the MRD is measured by quantitative detection of at least one of the cytogenetic abnormalities or transpositions selected from the group consisting of: t (12; 21) [TEL- AML1]; t (1; 19;) [E2A-PBX]; t (4; 11) [AF4-MLL]; t (9; 22) [BCR-ABL]; hyperdiploidy or trisomies of chromosomes 4, 10, and 17; hypodiploidy; transpositions into the immunoglobulin genes; and transpositions into the T cell receptor (TCR) genes. The construct for the use of any one of claims 1 to 6, wherein the corresponding variable regions of the (VH) chain and the corresponding light chain (VL) variable regions in the construction of said single chain bispecific antibody CD19xCD3 are arranged , from the N-terminal to the C-terminus, in the order VL (CD19) -VH (CD19) -VH (CD3) -VL (CD3). The construct for the use of claim 7, wherein the construction of said single chain bispecific antibody CD19xCD3 includes an amino acid sequence such as that described in SEQ ID NO. 1, or an amino acid sequence that is at least 90%, preferably 95%, identical to SEQ ID NO. 1. The construct for the use of any one of claims 1 to 8, wherein the pharmaceutical composition comprising the construction of the single chain bispecific antibody CD19xCD3 is to be administered by continuous infusion for at least four weeks, followed by a range of 2 weeks without treatment. The construct for the use of claim 9, wherein said administration is to be repeated at least two, three, four, five, six, seven, eight, nine or ten times after a negative state has been determined for MRD. The construct for the use of claim 9 or 10, wherein the method is applied prior to an allogeneic stem cell transplantation (HSCT), to transform a positive ALL for MRD, in a negative condition for MRD. The construct for the use of claim 9 or 10, wherein the method is applied after allogeneic stem cell (HSCT) transplantation. The construct for the use of claim 12, wherein the construction of the single chain bispecific antibody CD19xCD3 induces a grafting effect against leukemia (GvL). The construct for the use of any one of claims 1 to 13 wherein the construction of the single chain bispecific antibody CD19xCD3 is administered at a daily dose of 10 pg to 100 pg per square meter of body surface area of the patient. The construct for the use of any one of claims 1 to 13, wherein the construction of the single chain bispecific antibody CD19xCD3 is administered at a daily dose of 15æg to 30æg per square meter of body surface area of the patient. The construct for the use of any one of claims 1 to 13, wherein the construction of the single chain bispecific antibody CD19xCD3 is administered at a daily dose of 15æg, 30æg, 60æg, or 90æg per square meter of surface area of the patient's body. The construct for the use of any one of claims 1 to 13, wherein the construction of the single chain bispecific antibody CD19xCD3 is administered at a dose of 5æg on or on the first days, followed by administration of 15æg for the following days, followed by administration of 30 or 45 pg per square meter of body surface area of the patient as a daily dose over the remaining period of a total of 4 weeks. The construct for the use of claim 17, wherein the two starting doses are administered for 2, 3, 4, 5, 6, or 7 days, or even for a longer time. The construct for the use of claim 1 or 2, wherein said patient is ineligible for allogeneic stem cell transplantation. Lisbon, March 18, 2015.